COMPARATIVE EFFICACIES OF VARIOUS SANITIZERS USED IN
FOOD SERVICE ESTABLISHMENTS
A Thesis
Presented in Partial Fulfillment of the Requirements for
the Degree Master of Science in the
Graduate School of The Ohio State University
By
Gerald Sigua, B.S.
Graduate Program in Food Science and Technology
The Ohio State University
2009
Master’s Examination Committee:
Dr. Melvin A. Pascall, Advisor
Dr. Valente Alvarez
Dr. Ken Lee
ABSTRACT
Foodborne illness continues to be a major public health concern, especially due to
the improper washing and sanitization of tableware items in restaurants and foodservice
establishments. This could lead to cross-contamination of ready-to-eat foods and may
even lead to the formation of biofilms if inadequate washing becomes a long-term
practice.
As a result, cleaning and sanitization of all tableware items should be
maximized to minimize this risk. The FDA Food Code (2005) and NSF International
standards require a 5-log bacterial reduction in testing the efficacy of a chemical
sanitization method. These standards also mandate the complete removal of all food soil
from tableware items after the washing protocol. However, as a cost savings practice,
foodservice establishments usually wash several batches of tableware items in a single lot
of cleaning chemicals. As a result, there is a need to determine the maximum number of
warewashing cycles that can still achieve the Food Code and NSF standards, when a
single lot of detergent/rinse water/sanitizer is used.
The first study of this thesis (Chapter 2) evaluated the efficacies to two traditional
(sodium hypochlorite and quaternary ammonium) and two newer (neutral electrolyzedoxidizing water and PRO-SAN®) sanitizers. This was achieved by investigating the
survival of Escherichia coli K12 and Listeria innocua. These bacterial species were
inoculated into cream cheese and pasteurized whole milk and subsequently used to soil
ii
ceramic plates, plastic serving trays and drinking glasses. These tableware items were
washed with mechanical 49°C (120°F) and manual 43°C (110°F) dishwashers then
treated with one of the four sanitizers. After the washing protocol, viable bacterial counts
were determined on the surface of tableware items using the plate counting method, and
thus the maximum number of warewashing cycles were determined. Results showed that
the newer sanitizers are more efficient than the traditional sanitizers used in the
foodservice industry.
This study also concluded that mechanical washing is more
efficient than manual washing. Lastly, the type of material used to make the tableware
plays a role in the washing efficiency, as more glassware items were washed while still
achieving a 5-log bacterial reduction when compared with ceramic plates and plastic
trays.
The second part of this thesis (Chapter 3) investigated the effect of the various
sanitizers on removing various milk-based products (whole, 2% reduced fat, chocolate
low fat and skim milk) from underlining glass surfaces. Atomic force microscopy (AFM)
was used to determine the thicknesses of milk-films left after attempts to clean the glass
surfaces. These thickness measurements were then analyzed to determine the adhesion
potential between the residual milk samples and the glass surfaces. Results showed that
PRO-SAN® significantly reduced the amount of residual food soil when compared with
the other sanitizers. This was due to the presence of sodium dodecylbenzene sulfonate (a
surfactant) in the PRO-SAN® formulation.
The data also showed that whole and
chocolate milk would be more difficult to clean when compared with 2% and skim milks.
iii
These studies showed the comparative effectiveness of the novel sanitizers. This
is important because these newer sanitizers are less dangerous to workers and have a
lower negative impact on the environment. Information obtained from this study could
be used by restaurants and other foodservice institutions to minimize the cost of food
contact surface cleaning while still meeting FDA mandates.
iv
DEDICATION
Dedicated to my family
v
ACKNOWLEDGMENTS
First and foremost, I would like to show my sincere appreciation to my advisor,
Dr. Melvin Pascall. Completing this thesis would have not taken place had it not been for
his guidance and complete support throughout this two-year educational journey.
I
would also like to express my gratitude to Dr. Valente Alvarez and Dr. Ken Lee for their
help and for serving as part of my Master’s examination committee. Furthermore, I
would like to thank Dr. Gerald Frankel and Saikat Adhikari for their time and assistance
in completing my AFM research. I would like to also acknowledge and thank the Hobart
Corporation and CIFT for their help and financial support.
Additionally, I would like to show appreciation to my lab colleagues, Dr. Jaesung
Lee, Lizanel Feliciano-Sanchez and Aldo Handojo. Their knowledge and assistance in
this area of research were vital to the completion of my thesis. Additionally, they
provided the encouragement to finish my studies and more importantly became life-long
friends.
As a final acknowledgment, I would like to thank my family and close friends for
their love, guidance and support. My parents Gilbert and Celia and my younger sister
Christine, have supported me without hesitation during the writing of this journal.
vi
VITA
September 2, 1984……………………………..Born – Nueva Ecijia, Philippines
2001 – 2005……………………………………B.S. Food Science & Nutrition
University of Florida
Gainesville, FL
2005 – 2007……………………………………Research Microbiologist & Chemist
ABC Research Corporation
Gainesville, FL
2007 – Present…………………………............Graduate Research Associate
The Ohio State University
Columbus, OH
FIELDS OF STUDY
Major Field: Food Science and Nutrition
vii
TABLE OF CONTENTS
ABSTRACT ....................................................................................................................... ii
DEDICATION................................................................................................................... v
ACKNOWLEDGMENTS ............................................................................................... vi
VITA................................................................................................................................. vii
TABLE OF CONTENTS .............................................................................................. viii
LIST OF TABLES ........................................................................................................... xi
LIST OF FIGURES ....................................................................................................... xiv
CHAPTERS:
1. Literature Review............................................................................................................ 1
1.1. Introduction .............................................................................................................. 1
1.2. Foodborne Illness: Risk Factors............................................................................... 4
1.2.1. Consumer Knowledge ....................................................................................... 4
1.2.2 Foods ................................................................................................................. 5
1.2.3. “At Risk” Individuals ........................................................................................ 6
1.2.4. Food Handling Procedures ................................................................................ 6
1.3. Foodborne Microorganisms ..................................................................................... 9
1.3.1. Extrinsic Factors ............................................................................................. 10
1.3.2. Intrinsic Factors .............................................................................................. 13
1.4. Pathogenic Bacterial Foodborne Microorganisms of Concern .............................. 16
1.4.1. Listeria monocytogenes .................................................................................. 17
1.4.2. Escherichia coli .............................................................................................. 19
1.4.3. Salmonella....................................................................................................... 22
1.4.4. Staphyloccocus aureus .................................................................................... 23
1.4.5. Other Pathogenic Bacteria Associated With Foodborne Illness ..................... 24
1.5. Processing Methods To Control Bacterial Growth And Limit Survival Rates ...... 25
1.6. Tableware Washing And Sanitation In Food Establishments................................ 26
viii
1.7. Types Of Dishwashers ........................................................................................... 28
1.7.1. Mechanical Warewashers ............................................................................... 28
1.7.2. Manual Warewashers ...................................................................................... 30
1.8. The Importance Of A Cleaning And Sanitizing Program ...................................... 31
1.9. Cleaning Agents – Characteristics ......................................................................... 34
1.9.1. Atomic Force Microscopy .............................................................................. 36
1.10. Classes of Cleaning Detergents ........................................................................... 39
1.10.1. Soaps ............................................................................................................. 39
1.10.2. Alkaline Cleaning Agents ............................................................................. 40
1.10.3. Acidic Cleaning Agents ................................................................................ 41
1.10.5. Synthetic Detergents ..................................................................................... 42
1.11. Sanitizing Agents – Characteristics ..................................................................... 42
1.11.1. Chlorine......................................................................................................... 44
1.11.2. Quaternary Ammonium Compounds (QAC’s) ............................................. 47
1.11.3. Acidic-Based Sanitizers ................................................................................ 49
1.11.4. Neutral Electrolyzed-Oxidizing Water (NEW) ............................................ 50
1.11.5 Summary Of The Sanitizers Previously Discussed ....................................... 53
2. Comparative Efficacies Of Various Chemcial Santizers For Warewashing Operations
In Restaurants.................................................................................................................... 55
2.1. Abstract .................................................................................................................. 55
2.2. Introduction ............................................................................................................ 56
2.3. Materials and Methods ........................................................................................... 59
2.3.1. Preparation of Bacterial Cultures .................................................................... 59
2.3.2. Preparation of Food Samples .......................................................................... 60
2.3.3. Preparation Of Detergent And Sanitizer Solutions ......................................... 62
2.3.5. Manual Warewashing Of The Tableware Items ............................................. 67
2.3.4. Mechanical Warewashing Of The Tableware Items ....................................... 69
2.3.6 Microbial Enumeration Of The Contaminated Tableware Surfaces................ 71
2.3.7. Statistical Analysis .......................................................................................... 72
2.4. Results and Discussion .......................................................................................... 72
2.4.1. Effect Of Air-Drying On The Reduction Of The Microbial Population ........ 72
2.4.2. Efficacy Of The Various Sanitizing Agents Used In This Study ................... 73
2.4.3. Comparative Efficiencies Of Manual With Mechanical Warewashing
Operations ................................................................................................................. 77
2.4.4. Effect Of Material Type On The Efficiency Of Washing And Sanitizing
Protocols ................................................................................................................... 79
2.4.5. Survivability of Listeria innocua And Escherichia coli K12 After Washing
And Sanitizing Protocols .......................................................................................... 80
2.5. Conclusions ............................................................................................................ 81
ix
3. A Comparison Of Various Chemcial Sanitzers In The Removal Of Organic Matter On
Glass Surfaces By Atomic Force Microscopy .................................................................. 82
3.1. Abstract .................................................................................................................. 82
3.2. Introduction ............................................................................................................ 83
3.3. Materials and Methods ........................................................................................... 85
3.3.1. Food Sample Preparation ................................................................................ 85
3.3.2. Modified Washing Protocol ............................................................................ 86
3.3.3. Atomic Force Microscopy Measurements ...................................................... 87
3.3.4. Statistical Analysis .......................................................................................... 90
3.4. Results and Discussion .......................................................................................... 90
3.4.1. Force Required To Remove A Selected Milk-Based Film Area During The
AFM Scratching Test. ............................................................................................... 90
3.4.2. Milk-Based Film Thickness Data Of Traditional Sanitizers (Sodium
Hypochlorite And QAC) ........................................................................................... 93
3.4.3. Milk-Based Film Thickness Data Of Novel Sanitizers (EO-WATER and
PRO-SAN®) .............................................................................................................. 96
3.4.4. Confirmation Of AFM As A Reliable Analytical Procedure For Food Samples
................................................................................................................................. 101
3.5. Conclusions .......................................................................................................... 102
4. Conclusion .................................................................................................................. 103
List Of References .......................................................................................................... 105
APPENDIX A: RAW DATA ......................................................................................... 120
APPENDIX B: STATISTICAL ANALYSIS ................................................................. 131
x
LIST OF TABLES
Table
Page
1.1 Identifiable Instances When Hand Washing Is Mandatory…………….......................8
1.2. Unhygienic Practices of Food Handlers……………………………………………...9
1.3. Interaction Of pH And aw For Control Of Vegetative Bacteria And Spores
In Food Not Heat-Treated Or Heat-Treated But Not Packaged…………………………15
1.4. Common Foodborne Diseases………………………………………………………17
1.5. Minimum Washing And Sanitization Temperature Of Various
Hot-Water Sanitization Warewashing Machines………………………………………...30
1.6. Water Impurities And Associated Problems………………………………………...33
1.7. Characteristics of Food Soils………………………………………………..............35
1.8. Physical And Chemical Factors Affecting Sanitizer Effectiveness………...……….43
1.9. The Minimum Temperature Of Chlorine Sanitizing Solutions
For Food-Contact Surfaces At A Certain pH And Concentration…………………….....46
1.10. The Advantages And Disadvantages Of Chlorine-Based Sanitizers…..…..............46
1.11. The Advantages And Disadvantages of QAC’s………………………..…………..48
1.12. The Advantages And Disadvantages Of Acidic-Based Compounds……................50
1.13. Summary Of The Advantages And Disadvantages Of Chlorine,
QAC, Acidic-Based And EO Water Sanitizers…………………………………………..54
2.1. Bacteria Survival (log10 CFU/tableware) On Various Contaminated
Tableware Before And After 1 Hour Air-Drying At 24°C Prior To The Washing and
Sanitization Protocol…………...………………………………………………………...73
xi
3.1. The Force (V) Required To Complete Remove Various Milk-Based
Samples From The Glass Surfaces After Washing Protocols With Various
Sanitizers…………………………………………………………………………...…….91
A.1. Raw Data For The Maximum Number Of Warewashing Cycles That
Can Produce A 5-log Bacterial Reduction of Listeria innocua With A Single
Batch of Detergent, Rinse Water And Sanitizer……..…………………………………121
A.2. Raw Data For The Maximum Number of Warewashing Cycles That
Can Produce A 5-log Bacterial Reduction Of Escherichia coli K12 With A Single
Batch Of Detergent, Rinse Water And Sanitizer.………………………………………122
A.3. Raw Data For The Force Required To Remove A 5 x 5 µm Area Of Milk
Film From A Glass Surface After A Modified Washing Protocol With Sodium
Hypochlorite...………………………………………………………………………….123
A.4. Raw Data For The Force Required To Remove A 5 x 5 µm Area Of Milk
Film From A Glass Surface After A Modified Washing Protocol With QAC…...…….124
A.5. Raw Data For The Force Required To Remove A 5 x 5 µm Area Of Milk
Film From A Glass Surface After A Modified Washing Protocol With EO Water...….125
A.6. Raw Data For The Force Required To Remove A 5 x 5 µm Area Of Milk
Film From A Glass Surface After A Modified Washing Protocol With PRO-SAN®.....126
A.7. Raw Data Of Milk Film Thickness (nm) After A Modified Washing
Protocol With Sodium Hypochlorite……….…………………………………………..127
A.8. Raw Data Of Milk Film Thickness (nm) After A Modified Washing
Protocol With QAC………...…………………………………………………………...128
A.9. Raw Data Of Milk Film Thickness (nm) After A Modified Washing
Protocol With EO-Water……...………………………………………………………...129
A.10. Raw Data Of Milk Film Thickness (nm) After A Modified Washing
Protocol With PRO-SAN®…...…………………………………………………………130
B.1. Multifactorial ANOVA For Sanitizers Data For The Washing Experiment..……..132
B.2. Multiple Comparisons (Tukey HSD and Bonferroni) For Various
Sanitizers Used In The Washing Experiment...………………………………………...132
B.3. Multiple Comparisons (Tukey HSD and Bonferroni) For Various
Tableware Items Used In The Washing Experiment…...………………………………133
xii
B.4. Legend Used For AFM Statistical Analyses………………………...…………….134
B.5. Multiple Comparisons (Tukey HSD and Bonferroni) For Film
Thickness Of Various Milk Samples…………………………………………………...134
B.6. Multiple Comparisons (Tukey HSD and Bonferroni) For Various
Sanitizers Used In AFM Analyses…………………………….………………………..136
B.7. Multiple Comparisons (Tukey HSD and Bonferroni) For Whole
Milk-Film Thickness For Various Sanitizers Used In AFM Analyses………………....137
B.8. Multiple Comparisons (Tukey HSD and Bonferroni) For 2 Percent
Milk-Film Thickness For Various Sanitizers Used in AFM Analyses…………………138
B.9. Multiple Comparisons (Tukey HSD and Bonferroni) For Skim
Milk-Film Thickness For Various Sanitizers Used In AFM Analyses…………………139
B.10. Multiple Comparisons (Tukey HSD and Bonferroni) For
Chocolate Milk-Film Thickness For Various Sanitizers Used In AFM Analyses……...140
B.11. Multiple Comparisons (Tukey HSD and Bonferroni) For Film
Thickness With Sodium Hypochlorite And Various Milk-Types……………………...141
B.12. Multiple Comparisons (Tukey HSD and Bonferroni) For Film
Thickness With QAC And Various Milk-Types……………………………………….142
B.13. Multiple Comparisons (Tukey HSD and Bonferroni) For Film
Thickness With PRO-SAN® And Various Milk-Types………………………………...143
B.14. Multiple Comparisons (Tukey HSD and Bonferroni) For Film
Thickness With NEW And Various Milk-Types……………………………………….144
xiii
LIST OF FIGURES
Figure
Page
1.1. Diagram Of AFM Imaging Process………...……………………………………….37
1.2. Image Of A Cantilever And Tip…………………………………………..………...37
1.3 Anionic Surfactant Molecule……………………………………………………...…40
1.4 Soil Particle Suspended By Micelle Formation……………………………………...40
1.5. Chemical Structure Of QAC…………………………………………………...……47
1.6. The Schematic Process Of Electrolyzed Water Generator………………….............52
2.1. Tableware Used In Experimental Procedure (a) Ceramic Plates,
(b) Plastic Serving Trays And (c) Drinking Glasses……………………………………..60
2.2. Experimental Design Of Warewashing Procedure………………………………….63
2.3. EO Water Generator Used In This Study…………………………………...............65
2.4. Three-Compartment Turbowash Manual Warewasher Used In This Study………...67
2.5. Cylindrical Sponge (Left) And Sponge Attached To The Spring-Loaded
Test Fixture (Right) Used To Manually Clean Tableware Items………………………..69
2.6. AM Select Mechanical Washer Used In This Study………………..……………....70
2.7. The Maximum Number Of Warewashing Cycles That Can Produce A
5-Log Bacterial Reduction Of Listeria innocua With A Single Batch Of Detergent,
Rinse Water And Sanitizer…………………………………………………………...….74
xiv
2.8. The Maximum Number Of Warewashing Cycles That Can Produce
A 5-Log Bacterial Reduction Of Escherichia coli K12 With A Single Batch Of
Detergent, Rinse Water And Sanitizer………………………...…………………………75
3.1. An Experimental Replication Consisting Of 8 Microscope
Glass Slides (2 Slides With Either A Drop Of Whole Milk, 2% Reduced
Fat Milk, Chocolate Milk And Skim Milk)…………………………………………...…86
3.2. AFM Machine Used In This Experiment…………………………………………....88
3.3. Scanning Electron Microscope Microscope Image Of An AFM Tip Used To Probe
The Structure Of The Sample Surface (10,000x Magnification)….…….……………….89
3.4. A Tapping Mode 3-D Topographical AFM Image Of The Surface
Of A Whole Milk Sample After the Washing Protocol With Sodium Hypochlorite…....93
3.5. Cross-Sectional Analysis Of The Scratched Area Of The Whole Milk
Sample On The Glass Surface………...………………………………………………....94
3.6. Mean Thickness (nm) Measurements By AFM Analysis Of Various
Milk Products After Washing Protocols Using Sodium Hypochlorite…………………..95
3.7. Mean Thickness (nm) By AFM Analysis Of Various Milk Products
After Washing Protocols Using QAC…………………………………………………....96
3.8. Mean Thickness (nm) By AFM Analysis Of Various Milk Products
After Washing Protocols Using EO-Water……………………………………………....97
3.9. Mean Thickness (nm) By AFM Analysis Of Various Milk Products
After Washing Protocols Using PRO-SAN®………………………………………….…98
3.10. Mean Thickness (nm) By AFM Analysis Of Whole Milk After
Washing Protocols Using Various Sanitizers……………………………………………99
3.11. Mean Thickness (nm) By AFM Analysis Of Chocolate Milk After
Washing Protocols Using Various Sanitizers……………………………………………99
3.12. Mean Thickness (nm) By AFM Analysis Of 2 Percent Reduced Fat
Milk After Washing Protocols Using Various Sanitizers………………………………100
3.13. Mean Thickness (nm) By AFM Analysis Of Skim Milk After
Washing Protocols Using Various Sanitizers…………………………………………..100
xv
CHAPTER 1
LITERATURE REVIEW
1.1. Introduction
Foodborne illness continues to be a significant public health concern not only in
the United States, but also worldwide. Increased international trade and the increasing
number of individuals traveling abroad are the two major factors that increase the risk of
foodborne illnesses, despite the continual improvement in the quality and safety of food
production and handling (Kaferstein et al., 1997). According to Mead et al. (1999) there
are an estimated 76 million cases of foodborne illness, 325,000 hospitalizations, and
5,000 deaths that occur in the United States every year. The economic impact on the
individuals contracting these foodborne illnesses can be severe. Due to loss of individual
productivity, medical bills and the impact felt by the institution that the affected
individual works for, it is estimated that foodborne illnesses cost between $6.5 and $34.9
billion per year (Mead et al., 1999). If a company has to recall an unsafe product or it is
found that a national restaurant chain serves food in an unsanitary environment, there will
be a negative public perception of that company (Waites and Arbuthnott, 1990).
Furthermore, there can be possible legal action by the regulatory body with jurisdiction
1
over the company and by the affected consumers. This negative impact can be long-term
and it may take years, even decades, before the company or restaurant establishment can
regain consumer confidence. An example of this occurred when a corned beef typhoid
outbreak took place in 1964 in Scotland. It took this incident nearly 20 years before the
lost sales returned to pre-outbreak levels (Waites and Arbuthnott, 1990).
It is the goal of the Food and Drug Administration (FDA) and the United States
Department of Agriculture (USDA) to provide guidelines to food companies, restaurant
establishments and consumers on how to effectively minimize potential food hazards,
especially from pathogenic microorganisms and the toxins that they produce (Waites and
Arbuthnott, 1990). Microbial contamination of food can occur at every step of the food
chain, from production, processing, distribution, preparation and storage (Knabel, 1995).
Microorganisms are ubiquitous in nature and they are found in soil and on plants. Some
pathogens even exist on the skin and in the intestinal tracts of humans and animals
(Knabel, 1995). It is estimated that approximately 50% of food service workers that
handle food are contaminated with pathogenic microorganisms, although they may not
show signs or symptoms caused by the illnesses (Mariott & Gravani, 2006). It is thus
very difficult to completely prevent pathogenic contamination of the food supply.
A National Restaurant Association (2004) report states that the best approach to
prevent foodborne illness in restaurant establishments is to educate the food preparer,
server and dishware washer on ways to minimize contamination. This can be very
difficult, however, since most foodservice employees are young and do not stay
employed with the same restaurant for long periods of time.
Additionally, most
foodservice employees do not have good medical benefits and this means that they may
2
not get paid when they do not attend work. Thus, they are more likely to go to work
while having an illness (Jones and Angulo, 2006). Since it may sometimes be difficult
for management to detect a sick employee, this consequently increases the risk of crosscontamination from an ill employee who could shed pathogenic microorganisms into
prepared food.
If this is coupled with improper processing of the food or with
insufficient washing and sanitization of tableware, an increased risk of foodborne illness
could occur.
More than 13 million people work in the restaurant industry and there are over
945,000 foodservice establishments in the United States. This is a massive industry and
it is predicted that the 2009 sales forecast will be $566 billion (National Restaurant
Association, 2009). This equates to nearly 50% of all money spent in the United States
on any given day. It is estimated that approximately 44% of all adults eat at restaurants
on a given day (Jones et. al, 2004). According to Mariott & Gravani, 2006, the risk of
food-service related illness is approximately 1 in 9,000. This means that of the estimated
45 billion meals that are served in the United States annually, approximately 5 million
meals may be contaminated, and thus an enormous amount of food-borne illnesses can
potentially occur. Thus, careless errors in food preparation or insufficient sanitization of
food-contact surfaces can lead to several cases of illnesses due to the high amount of
customers that are being served each day (Jones and Angulo, 2006). What exacerbates
this problem is the fact that people who contract foodborne illness are more inclined to
accuse commercial establishments instead of blaming it on home cooked food or poor
personal hygiene. Although the exact percentage of foodborne illness that origintate
from the restaurant industry is not accurately known, the estimates clearly illustrate that
3
there exists the need to minimize potential food safety hazards. This can be done by
educating employees about the risk factors that are involved in foodborne illness.
1.2. Foodborne Illness: Risk Factors
1.2.1. Consumer Knowledge
According to a report by the Task Force of the Council for Agricultural Science
and Technology (CAST, 1994), a normal person may be uninformed that some
pathogenic microorganisms can grow at refrigeration temperatures. The report also said
that consumers have limited knowledge about the degree of bacterial contamination that
raw foods such as animal products, fresh fruits and vegetables may carry. This can also
be evidenced by a 2004 study performed by Redmond, et al., which observed the normal
behavior of individuals when preparing meals at home. While some individuals used a
wide variety of safe food preparation tactics, the majority of the observed participants
used unsafe food handling and cleaning protocols which could lead to an increased risk of
cross-contamination and foodborne disease.
It is thus essential that consumers are
educated about these things. An assortment of information on food safety are available to
consumers from government agencies (FDA, USDA, CDC, EPA, State Agencies), food
science departments at universities and colleges, food industry trade organizations,
consumer safety specialists and extension agents (Lin et al., 2005). It is therefore vital
that everyone that is involved in the food industry, whether it is food producers,
processors, servers, or dishware washers should be educated with the intent of preventing
foodborne illnesses (Medeiros et al., 2001).
4
1.2.2 Foods
A variety of foods are associated with foodborne illnesses, especially foods of
animal origin such as fish and shellfish, red meats and poultry. Fresh fruits, vegetables,
eggs, and dairy products could also be contaminated with pathogenic and spoilage
microorganisms (CAST, 1994).
Each type of food contains a certain nutritional
composition and has a common preparation and processing procedure. These specific
conditions create an excellent environment for specific spoilage and pathogenic
microorganisms to grow and be viable. For example, vegetables and fruits that is grown
on or close to the ground has a higher probability of being contaminated with sporeforming bacteria such as Bacillus cereus, Clostridium botulinum and Clostridium
perfringens (Bryan, 1988). These spores can survive severe environments such as the
presence of cleaning and sanitizing agents and various food processing techniques. Thus,
to ensure that they are receiving raw food with a low bacterial load, restaurants and
grocery stores need to make sure that safe growing, handling, and distribution practices
are implemented for produce and other fresh foods before they are used or sold to
consumers. This requires fruit and vegetable suppliers to implement Good Agricultural
Practices (GAP) and a phytosanitary plan according to the United States Environmental
Protection Agency (EPA, 2008). Foods that are acquired from unsanitary sources or of
unknown origin should not be considered safe and should not be used or sold to the
consuming public. Seafood obtained from sewage-polluted waters or wild mushrooms
are examples of foods that can be considered unsafe and should be categorized as
adulterated by the FDA or USDA (Knabel, 1995).
5
1.2.3. “At Risk” Individuals
Unsafe foods are especially hazardous to “at risk” individuals who may contract
illness at a higher rate than healthy persons.
At risk individuals include immuno-
compromised persons who are experiencing lengthy illnesses, the elderly, infants and
pregnant women (USDA, 2008). In the current demographic shift in the United States, a
majority of the baby-boomer generation is now heading into retirement. As a result, the
proportion of elderly individuals in the population is increasing. This elderly group is
more prone to illnesses and thus uses a higher proportion of medications, some of which
could further weaken their immune system. This group of the population should thus be
cognizant of their susceptibility to foodborne illnesses and for the need to educate
themselves about safe food handling procedures.
1.2.4. Food Handling Procedures
According to a report by the FDA National Retail Food Team (2004), over 900
food establishments were surveyed and several mishandling practices were found to be
“out of compliance”. Factors found to be out-of-compliance were improper holding
temperature and processing time of raw ingredients, specifically unprocessed raw ground
meat. This finding was also echoed by Knabel (1995) when he reported that one of the
leading causes of foodborne illness is insufficient cooking or heat processing of food.
This is so because raw animal products may contain pathogens and may possibly cause
illness if not properly cooked. For example, poultry usually contains various species of
Salmonella and Campylobacter, while ground beef may contain a variety of pathogens,
6
especially E. coli O157:H7 (Doyle, 2000). Additionally specific pathogens can also be
found on a variety of fresh fruits and vegetables. Some of these pathogens are capable of
surviving under harsh environmental conditions. Since many vegetables are not cooked,
great care must be taken to ensure that meals prepared from them have a low bacterial
load (Doyle et al., 1997). Furthermore, it is in the interest of consumers to know the
types of food that they order at restaurants so that they could avoid consumption of
“high-risk” foods, such as undercooked ground beef in hamburgers or “sunny side up”
eggs in which the yolk is not completely processed (Jones and Angulo, 2006).
Another important factor responsible for foodborne illness that was identified by a
large number of scientific research journals is cross-contamination within restaurants and
at the homes of consumers. Bacteria and other pathogens that are not properly removed
from food-contact surfaces, increase the risk of cross-contamination in freshly prepared
foods (Kusumaningrum et. al, 2002). In addition, another practice that can reduce the
risk of cross-contamination is the arrangement of the equipment in food processing plants
and at restaurants. An example of this arrangement is the placing of dishwashing and
waste equipment in locations that are far removed from the food preparation sites. This
arrangement decreases the potential for cross-contamination from dirty dishes,
contaminated air-flow and other avenues for outbreaks.
Furthermore, the poor personal hygiene of employees is a major safety hazard in
food establishments, particularly focusing on the hands as the primary means of crosscontamination (Snyder, 1998; Montville et. al, 2001; Bean et al., 1997).
Haysom and
Sharp (2005) performed a study about the bacterial contamination of domestic kitchens.
The results of this study showed that sites, such as the refrigerator handle and faucet tap,
7
that are repeatedly handled during the normal use of a kitchen, showed an increase in the
level of bacterial contamination. Thus, to reduce this risk, the washing of hands, wearing
of gloves and clean work attire will greatly minimize the risk of cross-contamination.
Table 1.1 displays the specific times when the washing of hands is mandatory, while
Table 1.2 shows other unhygienic practices by food handlers. A federal government
initiative, in conjunction with state governments and private companies, is helping to
reduce these food safety risk factors. This initiative is called the Healthy People 2010
and it establishes goals and challenges designed to ensure good health and longevity of
the population. One goal of this program is called the Food Safety Objective 10-6 and it
is designed to improve food employee behavioral practices that directly relate to
foodborne illnesses in retail food establishments. Healthy People 2020 is a similar
program that is currently being established by the National Network of Professionals
whose objectives include the promotion of a safe and healthy lifestyle for all consumers.
Table 1.1 Identifiable Instances When Hand Washing Is Mandatory (FDA Food Code,
2005; Schmidt and Rodrick, 2003).
•
•
•
•
•
•
Before beginning work
Before handling foods
After touching bare human body
parts other than clean hands and
clean arms
After using the restroom
After coughing sneezing, using a
tissue or handkerchief, using
tobacco, eating, or drinking
•
•
•
8
After handling soiled utensils or
equipment
During food preparation of raw
food products
When changing tasks where there is
a possibility for crosscontamination
When switching between raw and
cooked foods
Table 1.2. Unhygienic Practices Of Food Handlers (Schmidt and Rocdrick, 2003).
•
•
•
•
•
•
•
•
•
Wiping hands on clothes and aprons
Chewing gum and/or smoking
while working with foods
Holding toothpicks, straws, or other
objects in the month while
preparing foods
Wearing excessive jewelry on the
hands that may trap contamination
or fall into foods
Having long fingernails
•
•
•
•
Wearing nail polish
Wearing false fingernails
Touching pimples and boils
Not wearing water coverings over
bandages
Wearing soiled clothing
Touching the lip of a glass or the
end of a spoon
Handling money without washing
Not wearing a hair restraint
1.3. Foodborne Microorganisms
Reduction or elimination of foodborne microorganisms should be one of the most
important concerns for the food industry. This is so because microorganisms have the
ability to cause quality and spoilage losses and consequently a reduction in the profit
margin of food companies and restaurants. More importantly, certain microorganisms
are pathogenic and may cause mild to severe illnesses and even death in some cases.
Bacteria, viruses, fungi, parasitic protozoa, other parasites and marine phytoplankton are
the majority of organisms capable of causing foodborne illnesses (CDC, 2005).
There are two ways that microorganisms can cause illnesses: (1) infection and (2)
intoxication. Infection occurs when pathogens, which are in the food, are ingested and
subsequently grow in the intestines of the host (McSwane et al., 2005). Examples of
infectious pathogens are species of Escherichia O157:H7, Salmonella Enteritidis, Vibrio
cholerae and Shigella spp. (Schmidt et al., 2003). If the microorganisms stay within the
intestinal wall it is considered non-invasive, while those that enter the blood stream and
infect other tissues of the body are regarded as invasive microorganisms (Jay et al., 2005).
9
Intoxication, in contrast, arises when pathogenic microorganisms, such as Staphylococcus
aureus and Clostridium botulinum, produce a toxin in the food before it is ingested. To
cause an illness, the toxin level must reach a certain minimum concentration before
symptoms occur in the host. Depending on the strength of the host’s immune system, the
symptoms may be mild, severe or even lead to death (Hui et al., 2003). It is thus essential
to understand the dynamics of microorganisms, their method of replication and their
survival mechanisms.
This is so because extrinsic and intrinsic factors, which are
interrelated, play a large role in the growth and viability of spoilage and pathogenic
microorganisms.
1.3.1. Extrinsic Factors
Extrinsic parameters are defined as the properties of the environment in which the
food and contaminating microorganisms are stored. Extrinsic factors include storage
temperature, relative humidity, the presence of other microorganisms, the chemical
environment and gaseous atmosphere (Sockett, 1995).
The most important factor that can be controlled by the food industry is storage
temperature. Microorganisms grow over a broad range of temperatures, but they are
known to have an optimal range for rapid growth. For a given food processor, it is thus
vital to know the characteristic of the microorganisms of concern. This will depend on
the type of food being processed.
Once these microorganisms are identified, their
optimal growth temperature range can be recognized and growth can be minimized by
setting the temperature outside of this optimal zone. There are three general classes in
10
which microorganisms are categorized: (1) psychrotrophs, (2) mesophiles and (3)
thermophiles (Lee et al. 2007). Psychrotrophs grow well at or below 7°C, with an
optimum growth between 20-30°C. Mesophiles have an optimum range of 20-45°C and
thermophiles grow well at or above 45°C (Troller, 1993).
Another extrinsic factor that the industry must consider is the relative humidity of
the storage atmosphere. This aspect is important since it relates to the water activity (aw)
of the food. Since a direct relationship exists between relative humidity and temperature,
this should be considered when storing food products. As a general rule, the higher the
temperature, the more moisture the air can hold (Ray. 2004). Furthermore, foods with a
high aw tend to lose moisture when stored in an atmosphere of low relative humidity. On
the other hand, low aw foods pick up moisture in conditions of high relative humidity.
When the surface of a food picks up moisture, the aw increases and consequently become
conducive to microbial growth. This will eventually lead to growth within the matrix of
the food product and this could lead to a major quality and/or safety issue. When
moisture is lost from a foods surface the lower aw will lower the risk of microbial growth,
but it may make the food unacceptable in terms of its organoleptic properties.
The presence of other microorganisms is another factor that plays a role in the
growth or deactivation of specific spoilage and pathogenic microorganisms. This is so
because microorganisms compete for nutrients (Jay et al., 2005). This competition for
nutrients naturally selects for stronger microorganisms that can better survive the existing
environment. Also, certain microorganisms may naturally excrete metabolic waste that
could be lethal or inhibit the growth of other microbes. One example is Lactic Acid
Bacteria (LAB) which excrete acidic metabolic waste that may inactive acid-sensitive
11
bacteria (Ray, 2004). Other examples of inhibitory substances that are excreted by
microorganisms to inhibit the growth of other microbes include bacteriocins, hydrogen
peroxide and antibiotics. It is estimated that 30-99% of all bacteria produce at least one
bacteriocin to assist in inactivating other microorganisms present in the food matrix
(Cotter, et al., 2005).
Another important extrinsic factor is the presence and concentration of
atmospheric gases in the environment. Oxygen (O2) concentration is the most important
atmospheric gas. There are three classes of microorganisms that are influenced by the
presence of oxygen: (1) aerobes, (2) anaerobes and (3) facultative anaerobes (Mariott and
Gravani, 2006). Aerobic microorganisms, like Pseudomonas, require the presence of
oxygen, while anaerobic microorganisms (Clostridium species) require the complete
absence of oxygen in the environment. The facultative class has the ability to grow in the
presence or absence of oxygen, but would prefer environments with oxygen.
In addition to O2, Carbon Dioxide (CO2) is another important atmospheric gas
that is primarily used to control microorganisms in food products.
This is called
modified atmosphere packaging (MAP). In this system, the atmosphere of the package is
deliberately modified by decreasing O2 and increasing CO2 concentrations in order to
inhibit spoilage and pathogenic agents (Chruch and Parsons, 1995). This prolongs the
shelf-life of fresh-cut produce and improves the appearance of fresh meats and seafood
(Luo, 2007). O2 levels are limited to inhibit the growth of aerobic microorganisms while
simultaneously having enough O2 to prevent anaerobic respiration. CO2 is added as a
bacterial and fungal growth inhibitor (Dixon and Kell, 1989). The mode of inhibition by
CO2 is the disruption on enzymatic decarboxylations and the interference with the
12
permeability of cell membranes (King and Nagel, 1975; Enfors and Molin, 1978). MAP
is particularly useful for fresh meats, with high levels of CO2 (25-100%) providing a
longer shelf-life with the retention of the natural red meat color (Gill and Penny, 1988).
MAP works particularly well in combination with a low storage temperature, especially
with seafood products. Studies have shown that an optimal CO2 range for fish is 40-50%
and stored at refrigeration temperatures can double the shelf-life of the product as
compared conventional packaging (Sivertsvik et al., 2002).
1.3.2. Intrinsic Factors
Intrinsic factors are those parameters that are inherent in the microorganism itself.
These factors can not be controlled by the food industry and it is therefore essential to
recognize these parameters and place or subject the microorganisms in question to
conditions that hinder their growth and their pathogenic actions. The most important
intrinsic parameters include pH, moisture content (water activity), oxidation-reduction
potential (Eh), and nutrient requirements (Beuchat, 2002).
Most microorganisms have optimal growth in a neutral environment (a pH range
of 6.6-7.5) (Corlett and Brown, 1980; Jay et al., 2005). However, some may be able to
grow at pH levels as low as 4.6 and as high as 8.0 (Troller, 1993). The pH value of 4.6 is
a significant value in the food industry. This numerical marker defines if a food product
is low-acid (pH 4.6 and greater) or acidic (below pH 4.6) (Blackburn, 2006). Acidic
foods are capable of denaturing the cell membranes of most bacteria, and have thus a
lower initial microbial load when compared with low acidic foods. A lower microbial
13
load decreases the processing time and this allows the product to retain more nutrients,
improves the final quality of the product and may also increase its shelf-life. However,
most molds and yeasts are capable of surviving an acidic environment and this explains
why most fruits (which are generally acidic) are more prone to mold and yeast when
compared with bacterial growth (Corlett and Brown, 1980). Fresh meats, of any origin,
are usually at or near a neutral pH and this make them more susceptible to microbial
growth especially if temperature abused (Blickstad and Molin, 1983).
The moisture content of a food also plays an essential role in the growth and
activity of microorganisms. The amount of moisture in a food is based on the ratio of
free and bound water molecules (Mathlouthi, 2001). Bound water molecules are not
available for use by microorganisms that may be present in the food. This is so because
bound water molecules are chemically bonded to large food molecules and cannot
participate in any other chemical or metabolic reactions. Free water molecules, however,
can be readily used by microorganisms for essential functions, especially for growth and
replication (Jay et al., 2005). This relationship of free versus bound water is related to its
water activity (aw) value, which is currently being used in the industry to describe the
water requirements of microorganisms. This parameter (aw) is defined as the ratio of the
water vapor pressure of the food (p) to the vapor pressure of pure water (p0) at the same
temperature: aw= p/p0 (Troller, 1986). Generally, most bacteria need a aw of at least 0.90,
while most yeast and molds require a aw of at least 0.8 (Grant, 2004). Table 1.3 displays
the interaction of the pH and the aw and how it affects the growth and survival of
microorganisms in ready-to-eat food. Certain aw values combined with specific pH
values work well in controlling growth, while other aw and pH combinations create a
14
potentially hazardous food (PHF). This PHF is defined in the FDA Food Code (2005) as
one that requires time/temperature control for its safety (TCS) to limit the growth of
pathogenic microorganism or the formation of bacterial toxins.
Table 1.3. Interaction Of pH And aw For Control Of Vegetative Bacteria And Spores In
Food Not Heat-Treated Or Heat-Treated But Not Packaged
Ph Values
aw Values
< 4.2
4.2-4.6
Non-PHF/NonNona
TCS Food
PHF /NonTCSb food
0.88-0.90
Non-PHF/Non- Non-PHF/NonTCS Food
TCS Food
> 0.90-0.92
Non-PHF/Non- Non-PHF/NonTCS Food
TCS Food
> 0.92
Non-PHF/NonPA
TCS Food
a
PHF – Potentially hazardous food
b
TCS - Time/Temperature Control for Safety Food
c
PA – Product Assessment Required
< 0.88
> 4.6-5.0
> 5.0
Non-PHF/NonTCS Food
Non-PHF/NonTCS Food
Non-PHF/NonTCS Food
PA
PAc
PA
PA
PA
Oxidation-reduction potential (Eh) is also an important parameter that affects the
optimum growth of microorganisms. Eh is defined as the difficulty or the ease in which a
substrate loses or gains electrons (Schmidt and Rodrick, 2003). When a substrate gains
an electron, the substrate is considered reduced, and when a compound loses an electron
the substrate is oxidized. Certain microorganisms require reduced conditions (low Eh
value) while others need an oxidized environment (high Eh value). A low Eh will favor
the growth of anaerobic microorganisms, while aerobic microorganisms will grow readily
15
under a high Eh value potential (Marriott, 2006). Facultative microorganisms are capable
of growth under either potential conditions.
Finally, another important intrinsic character is the nutrient requirements of
microorganisms. In order to grow, replicate and perform necessary metabolic functions,
an energy source is required. Sources of energy utilized by microorganisms are sugars,
alcohols and amino acids (Ray, 2004). Another nutrient need is nitrogen, which is
primarily found in amino acids, peptides and proteins. Additionally, microorganisms
require small amounts of vitamins (especially B vitamins) and minerals for survival.
Gram-positive bacteria can not synthesize these vitamins and minerals, while most gramnegative bacteria and molds are capable of synthesizing most or all of their vitamins and
minerals. For this reason, most gram-positive bacteria will be found growing in foods
that have high concentrations of B-vitamins (Jay et al., 2005).
1.4. Pathogenic Bacterial Foodborne Microorganisms of Concern
Illnesses can be contracted via a person-to-person transmission, an unsanitary
environment, or from contaminated foods.
Although approximately 30% of all
foodborne illnesses are associated with bacterial agents, bacteria cause nearly 75% of all
deaths (Mead et al., 1999). It is therefore important to be knowledgeable of known
pathogens that can be associated with the consumption of contaminated food. This is
displayed in Table 1.4.
16
Table 1.4. Common Foodborne Diseases (Clive, 1993).
Disease
Duration
Symptoms
Typical Foods
Listeriosis (Listeria
monocytogenes)
3-70
Days
Raw milk, cheese
and vegetables
Enterohemorrahagic
infection (E. coli)
2-9 days
Meningocephalitis;
stillbirths;
septicemia/meningitis
in newborns
Watery, bloody
diarrhea
Salmonellosis
(Salmonella
species)
1-4 days
Staphylococcal
food poisoning
6-24 hrs
Bacillus cereus
Food Poisoning
12-24 hrs
Diarrhea, cramps,
vomiting
Shigellosis
(Shigella species)
4-7 days
Diarrhea, fever,
nausea, vomiting,
cramps
Raw Foods
Human fecal
contamination
Streptococcal
foodborne infection
Clostridium
perfringens food
poisoning
1-3 days
Sore throat, scarlet
fever
Diarrhea, cramps,
rarely nausea and
vomiting
Raw milk,
deviled eggs
Cooked meat and
poultry
Handlers with
sore throats
Soil, raw foods
Diarrhea, cramps,
nausea vomiting,
fever, headache
Fish and seafood
Marine coastal
environment
Vibrio
parahaemolyticus
foodborne infection
12-24 hrs
4-7 days
Diarrhea, abdominal
pain, chills, fever,
vomiting,
dehydration
Nausea, vomiting,
diarrhea, cramps
Raw/undercooked
beef, raw milk
Raw,
undercooked
eggs; raw milk,
meat & poultry
Ham, meat,
poultry, creamfilled pastries,
cheese
Meat products,
soups, sauces,
vegetables
Mode of
Contamination
Soil or infected
animals,
directly or via
manure
Infected cattle
Infected foodsource animals;
human feces
Handlers w/
colds, sore
throats, infected
cuts
From soil or
dust
Prevention
of Disease
Pasteurization
of milk,
adequate
cooking
Cook beef
thoroughly,
pasteurize
milk
Cook eggs,
meat &
poultry
thoroughly
Thorough
heating; rapid
cooling of
foods
Thorough
heating; rapid
cooling of
foods
General
sanitation;
cook foods
thoroughly
General
sanitation
Thorough
heating; rapid
cooling of
foods
Cook
fish/seafood
thoroughly
1.4.1. Listeria monocytogenes
Listeria is a gram-positive, rod-shaped microorganism which displays a
distinctive movement called tumbling motility propelled by their peritrichous flagellum
(Ferreira et al., 2003). This microorganism is characterized by being oxidase-negative,
catalase-positive, and ferments glucose with production of acid without gas (Varnam and
Evans, 1991). Of the several known species of Listeria, only L. monocytogenes is
17
frequently associated with illness in humans, especially through consumption of foods. L.
monocytogenes is found naturally in the environment, especially in the soil, the intestinal
tracts of a variety of animals and humans, sewage and feces (Doyle, 1985; Blackman and
Frank 1996). L. monocytogenes has been implicated in fresh and frozen meat, poultry,
seafood, ready-to-eat products, unpasteurized milk and other dairy products, especially
cheeses (Kim and Frank 1995). If raw meats are not properly processed and/or the
surfaces of processing equipment and utensils are not properly cleaned and sanitized,
there can be a high risk of cross-contamination that could lead to outbreaks.
L. monocytogenes has 13 known serotypes and illness can be caused by any of the
13, with ½a, ½b and 4b being the three serotypes that cause the most foodborne illness
(Farber and Peterkin, 2000). These organisms are capable of growing at refrigeration
temperatures and as high as 45°C, with an optimal temperature range of 30-37°C (Frank
and Koff, 1990). This shows that L. monocytogenes is very dangerous because it can
survive refrigeration temperatures. Survival and growth of L. monocytogenes cells can
occur in an environment of pH 4.39-9.4 and also requires an aw is at least 0.92 (Hui et al.,
2003). A characteristic that distinguishes L. monocytogenes from other vegetative
bacteria is its high salt-tolerance level, and it is known to survive in sodium chloride
(NaCl) levels of up to 30% (Gellin and Broome, 1989).
An outbreak caused by L. monocytogenes can be observed usually between 1-2
weeks after the ingestion of contaminated food. The symptoms are “flu-like” with
diarrhea and a mild fever and in more sever cases the results could be meningitis,
miscarriage and perinatal septicemia (Knabel, 1995).
Most healthy individuals,
especially those with strong immune systems, rarely exhibit symptoms caused by this
18
pathogen (Russell, 1997). This pathogen is considered “opportunistic” as it mostly
affects those that are immuno-compormised, the elderly, infants and particularly pregnant
women. Duxbury (2004) reported that pregnant women are approximately 20 times more
vulnerable for contraction of the illness Listeriosis.
It has also been reported that
Listeriosis causes an estimated 2,500 illnesses and 500 deaths in the United States
annually (Hui et al., 2003). Although the number of cases is considerably under-reported,
the ratio of deaths to the total number of reported illnesses signifies that this pathogen is
considered very dangerous to “high-risk” individuals.
1.4.2. Escherichia coli
Along with Salmonella and Shigella, the genus Escherichia is part of a large
family called Enterobacteriaceae. Like most members of the Family Enterobacteriaceae,
Escherichia coli are gram-negative, rod-shaped facultative anaerobes with peritrichous
flagella. They are able to ferment sugars which produce lactic acids and other products
and have the ability to reduce nitrates (NO3-) to nitrites (NO2-) (Park et al., 1999). The
optimal conditions for growth include a temperature range of 7-48°C with an optimum of
37°C, a pH range of 4.4-9.0 and a minimum aw of 0.95 (Varnam and Evans, 1991).
These microorganisms are found in warm-blooded organisms and particularly colonize
the gastrointestinal tract of a human infant within 48 hours of birth (Neill et al., 2001).
Most E. coli strains are harmless, but 5 of them are virulent.
These five strains are (1) Enteroadherent-aggregative (EA-AggEC), (2)
Enterotoxigenic (ETEC), (3) Enteroinvasive (EIEC), (4) Enteropathogenic (EPEC) and (5)
Enterohemorrahagic (EHEC).
Each of these have distinctive characteristics in its
19
pathogenicity and are further classified by serotyping based on the K antigen (capsular;
heat labile somatic), the O antigen (lipopolysaccharide; heat stable somatic), and the H
antigen (flagellar) (Schmidt and Rodrick, 2003). These strains are most commonly found
in the feces of animals and may contaminate raw meat, especially if the processing area is
unsanitary and littered with feces. Infection from any of these 5 strains of E. coli can
cause urinary tract infections (UTI), gastroenteritis, infant meningitis and in rare but
severe cases, hemolytic-uremic syndrome (HUS) (Line et al., 1991).
Both the EA-AggEC and ETEC groups are named based on the characteristic
aggregation of body tissue cells by the binding of their fimbriae to the inside of the
intestinal lumen of the host (Kaper, J.B., 2005). Once attached to the intestinal wall,
ETEC plasmids encode the production of heat-stable toxins (ST) and heat-labile toxins
(LT). EA-AggEC also produces hemolysin exotoxins, which causes the lysis of red
blood cells, and heat-stable toxins (ST) which is similar to the one produced by the
ETEC group (Doyle et al., 1997). These exotoxins are the cause of watery diarrhea,
abdominal pain and sometimes vomiting (Nataro and Kaper, 1998). Due to the loss of
fluids and electrolytes in the body, dehydration is common and may even lead to death if
proper rehydration is delayed. According to the World Health Organization (2009), it is
estimated that ETEC causes more than 200 million cases each year, with between
170,000 to 380,000 of those cases causing death, mostly in developing and third world
countries. ETEC is also associated with the phenomenon knows as “travelers’ diarrhea”
which is the rapid onset of foodborne illness in individuals from industrialized countries
who travel to developing countries. Inadequately treated water in developing countries
20
and foods like turkey, salad vegetables, and seafood in industrialized countries are
associated with ETEC (Hui et al., 2003).
Both EIEC and EPEC are similar with regards to the facts that their pathogenic
mode of action and symptoms closely resemble the illness caused by Shigella spp.
(Varnam and Evans, 1991). Once they enter the host, colonization of the intestinal
epithelial cells begins, then replication and finally spreading into adjacent cells and
tissues occurs (Jay et al., 2005). EPEC is lacks fimbriae, and does not produce ST and
LT toxins and is moderately invasive. EIEC, on the other hand, is highly invasive. The
onset of symptoms usually occurs between 12-36 hours after infection with EPEC, with
symptoms being a watery diarrhea. EIEC infections produces bloody stools and may
sometimes produce a high fever (Hui et al., 2003).
The last class of pathogenic E. coli is EHEC. All EHEC strains produce Shiga
toxin 1 and/or Shiga toxin 2, which is also known as verotoxin 1 and verotoxin 2,
respectively (Paton and Paton, 1998). E. coli O157:H7 is the most common strain
associated with foodborne illness, with outbreaks associated with foods such as ground
beef, dry-cured salami, lettuce, spouts and unpasteurized fruit juices and raw milk (Mead
et al., 1999; Keene et al., 1997). The O157:H7 strain is distinctive from the other EHEC
strains due to the infectious dose being very low (2,000 cells or less), due to its tolerance
of acid environments and low temperatures (Leyer et al., 1995). The onset of symptoms
occurs 12-60 hours after consumption, starting with mild, non-bloody diarrhea, mild
abdominal pain and fever. As the microorganisms continue to replicate, the abdominal
pain and fever become severe, with bloody diarrhea (Sartz et al., 2008). Infection can
become even more severe with the development of hemolytic uremic syndrome (HUS),
21
which most commonly occurs in children 10 years and younger (Griffin and Tauxe, 1991;
Buchanan and Doyle, 1997). The HUS condition causes a number of complications,
including renal failure, seizures, coma and pancreatitis.
1.4.3. Salmonella
The Salmonella genus is characterized by being gram-negative, oxidase-negative,
rod-shaped and has peritrichous flagella, which project in all directions (Varnam and
Evans, 1991). There are over 2,000 serotypes of Salmonella, with serotype Typhimurium
and serotype Enteritidis being the most prevalent in the United States Salmonella
generally grow at a temperature range of 5-47°C, aw of 0.86 or higher and a pH range of
3.6 to 9.5 (FDA, 2008). The infective dose for Salmonella to cause illness in healthy
people depends on the serovar in question. For example, serovar Thyphimurium requires
a high number of cells to be ingested than the Typhi serovar (Cary et al., 2000; Blaser
and Newman, 1982).
Salmonellosis typically occurs 12-72 hours after infection and the duration of
illness can be from 4-7 days (FDA, 2008). Typical symptoms are nausea, vomiting,
diarrhea, abdominal cramps and fever (Goodridge et al., 2003). The mortality rate of this
pathogen is very low, with most deaths occurring in the usual “at risk” population. With
most Salmonella serotypes living in the intestinal tracts of animals, especially birds, the
most common contaminated foods are raw poultry, underprocessed poultry, eggs
produced by infected animals, cross-contaminated fruits and vegetables and more
recently with peanut-based food products (FDA, 2009). The Centers for Disease Control
and Prevention (CDC) (2008) reported that an estimated 1.4 million cases, 15,000
22
hospitalizations and 500 deaths occur every year in the United States. Additionally, the
Foodborne Diseases Active Surveillance Network (CDC - FoodNet) (2004) of the CDC
concluded that Salmonella is responsible for the largest number of foodborne illnesses in
the United States. This statistic signifies that proper handling and processing of poultry
products should be between 68°C - 72°C (145°F - 160°F) to ensure that the food is free
of Salmonella.
1.4.4. Staphyloccocus aureus
Genus Staphylococcus is part of the family Micrococcaceae and characterized as
being a facultative anaerobe, gram-positive, cocci-shaped, catalase-positive, grows in
clusters and can metabolize glucose. Conditions for growth include a temperature range
of 7-48°C, a aw as low as 0.86 and a survivability in an environment with 20% salt
concentration (Baird-Parker, 2000).
There are more than 20 identified species of Staphylococcus, with S. aureus being
the major cause of foodborne illness. S. aureus is ubiquitous and can be found on human
skin, hair, the nasal cavity, wounds, the air and can survive on clothing for long periods
of time (Doyle et. al, 1997). Due to its wide distribuition in the environment and being a
poor competitor to other microflora, post-processing contamination is the greatest source
of contamination, especially in ready-to-eat foods that are cross-contaminated by
inidivuals who are shedding these cells. Another cause of S. aureus food poisoning is
due to temperature abuse (Rode et al., 2007).
The main pathogenic action is the production of nine types staphylococcal
enterotoxins (SE) which is produced in the food and subsequently ingested by the host.
23
This is thus called a food intoxication (Balaban and Rasooly, 2000; Soriano et al., 2002).
The nine SE’s are designated A, B, C1, C2, C3, D, E, F and G, with types A and D being
the major culprits for illness (Mossel et al., 1995, Dinges et al., 2000). These SE’s are
responsible for inflammation of the intestines and the stomach, and is also known as
gastroenteritis.
The common acute symptoms are nausea, vomiting and abdominal
cramps, and these begin in less than 6 and up to 10 hours after ingestion of the toxin
(Murphy et. al, 2009). This toxin eventually leads to infections of the skin (boils,
cellulitis, and impetigo) and can cause more serious infections like pneumonia,
meningitis and abscesses of muscle, urogenital tract, central nervous system, and other
abdominal organs (Murray et al., 1999; Lund et al., 2000).
1.4.5. Other Pathogenic Bacteria Associated With Foodborne Illness
There are many more identifiable and known pathogenic microorganisms that can
cause foodborne diseases. Each microorganism has its own preference in the type of
food they inhabit and has optimal environmental factors that assist them in growth,
replication and virulence (CFSAN, 1998). An example of such a pathogenic bacteria is
the genus Vibrio and specifically the species (1) V. cholerae, (2) V. parahaemolyticus and
(3) V. vulnificus (Hall, 1997; Kaper et al., 1995). These species typically grow on
shellfish, contaminated water and produce the typical foodborne illness symptoms, such
as nausea, vomiting and diarrhea. Other microorganisms of concern include Clostridium,
Yersinia, Shigella spp., and many types of protozoa and viruses. Norwalk viruses for
example causes a majority of foodborne illnesses, but are rarely diagnosed due to
symptoms dissipating within 48 hours (CDC, 2005).
24
1.5. Processing Methods To Control Bacterial Growth And Limit Survival Rates
Ensuring microbial safety and extending the shelf life of a food product are the
primary objectives of all food processing companies and retail food service businesses.
The traditional preservation method has been the use of heat, and has served the food
industry well for more than a century. Processing techniques that require the use of heat
include retorting, pasteurization, aseptic processing, dry heat, and wet heating methods
such as blanching, and boiling (Fellows, 2005). Most microorganisms that grow in food
and cause foodborne illness thrive in the temperature danger zone. This danger zone
includes temperatures between 7°C-60°C (45°F-140°F) (Barbosa-Canovas and Gould,
2002). In order to eliminate and prevent growth of these target microorganisms, heating
should be applied and held at a temperature above this danger zone for a certain amount
of time depending on the type of food that is being processed.
Once adequately
performed, these heat treatment methods have shown the ability to kill microorganisms,
deactivate harmful enzymes and transform indigestible food into palatable products
(Montville and Matthews, 2005). However, these methods have the potential to reduce
the nutritive value, natural texture, color, taste and aroma of foods and reduce the quality
perceptions of consumers. Additionally, recent changes in consumer preference have
shifted from traditional highly processed foods with many added preservatives, to fresher
and minimally processed food products. Thus, any technology that processes foods
without microbial and or chemical contamination but minimizes the loss of its natural
characteristics is a tremendous advantage to the food industry (Smelt, 1998).
In response to this health-conscious revolution, food scientists and engineers have
been researching and developing novel non-thermal preservation methods. These new
25
processing technologies have minimized the use of heat, while still being able to ensure
safety of the food product. Some methods include high pressure processing, pulsed
electric field, ozone, irradiation and electrolysis of water (Ozen and Floros, 2001). Some
of these techniques are sill in the developmental stages and more research is need before
they are commercialized.
1.6. Tableware Washing And Sanitation In Food Establishments
With over 45 billion meals served per year in the United States, there is a very
high probability that illness can be caused by a few batches of improperly cleaned
tableware. Tableware is defined by the FDA Food Code (2005) as any eating, drinking,
and serving utensil; including flatware (forks, knives, and spoons), hollowware (bowls,
cups, serving dishes) and plates. The 2004 report by the FDA National Food Retail Team
concluded that inadequate cleaning and sanitization of food-contact surfaces was the
most common “out of compliance” procedure in the over 900 facilities that were
observed. During normal business hours in restaurants for example, multi-use items
become soiled during consumer use and must be washed and sanitized. Additionally,
food processing equipment and warewashing equipment also become soiled and require
ongoing cleaning and sanitization. This component of the food establishment business is
therefore vital in preventing illness and causing undue hardship to consumers.
26
As stated by Mariott and Gravani (2006), the largest cost of a cleaning and
sanitization program is labor, with nearly 50 cents of the sanitation dollar being spent on
hiring dishwasher employees and quality assurance personnel. The employee assigned as
the primary dishwasher is specifically vital to any sanitation program. He or she must be
familiar with any and all suggested and mandatory procedures. Any minor error or the
skipping of required washing steps increases the risk of tableware, food preparation and
warewashing equipment contamination. This is especially important when bacteria are
transferred to ready-to-eat foods (RTE) (Guzewich and Ross, 1999). Many RTE foods
are usually sealed in some type of packaging and are opened immediately before placing
them on tableware items. If the tableware is contaminated due to insufficient cleaning,
the bacteria can subsequently be transferred to the food and ingested by customers.
Furthermore, most commercial dishwashers require the need for trained employees to
correctly operate them and this, together with repair and maintenance costs could be
expensive.
Despite the high cost of purchasing high-quality food and warewashing
equipment, cleaning compounds, sanitizers and a labor workforce, the minimization of
foodborne disease risk factors greatly outweighs the consequences of an unsanitary and
inadequate sanitation program.
Choosing the appropriate warewashing equipment,
suitable cleaning compounds and optimal sanitizers will make the process more efficient
and in the long term be economically sound. However, inappropriate use of cleaning
compounds and sanitizers costs the food industry millions of dollars because litigations
and possible negative publicity after random regulatory inspections could deem the
operation inefficient (Mariott & Gravani, 2006).
27
1.7. Types Of Dishwashers
There are two classes of dishwashers that are available: (1) mechanical and (2)
manual three-compartment sink (Verran et al., 2000). The materials that are used to
create these dishwashers must not permit the migration of harmful substances or impart
colors, odors, or foreign tastes to the food (FDA Food Code, 2005). Additionally, the
equipment or machine must be durable to withstand repeated warewashing, must be
corrosion-resistant, have a finished to have a smooth, easily cleanable surface and
resistant to (1) pitting, (2) chipping, (3) scratching, (4) distortion and (5) decomposition.
1.7.1. Mechanical Warewashers
The biggest advantage of using a mechanical dish washer is that there will always
be uniform cleaning conditions, provided that the machine is not defective. On the other
hand, manual washing equipment requires the use of employees who could be variable in
their washing styles. Sometimes, when using a three-compartment sink protocol, a batch
of soiled tableware could be cleaned thoroughly, while other batches could be
inadequately cleaned. Another benefit is the higher throughput of mechanical machines
when compared with manual operations. When compared to manual warewashing sinks,
mechanical washers are expensive, must be maintained, with the possibility of extra costs
for repair.
According to the 2005 FDA Food Code (4-204.113) all mechanical warewashing
machines must have an accessible and readable data plate that is attached on the machine.
This data plate must specify the machine’s design and operation specifications including:
(1) Temperatures required for washing, rinsing, and sanitizing;
28
(2) Pressure required for the fresh water sanitizing rinse unless the machine is
designed to use only a pumped sanitizing rinse; and
(3) Conveyer speed for continuous machines or cycle time for stationary rack
machines.
This is an important requirement because it gives a quick and visible reference of the
standard operating procedures of that particular mechanical machine.
There are two types of mechanical washers: (1) those that use chemical sanitizers
and (2) those that use hot water as a sanitizer. For those machines that use chemical
sanitizers, a wash temperature of 49°C (120°F) is recommended, and the sanitizer must
be at a temperature that is required by the manufacturer. For example, the 2005 Food
Code states that the commonly used chemical chlorine sanitizers require a temperature of
24°C (75°F). Moreover, it is necessary for the machine to subject the tableware being
washed to the appropriate chemical sanitizer, at the required concentration and for the
desired contact time (Schmidt and Rodrick, 2003).
For those machines that use hot water as a sanitizer, the minimum temperature
that must be achieved on the surface of tableware is 65°C (150°F). There are four types
of mechanical hot water sanitizing machines (see Table 1.5), each with specific washing
solution temperature and sanitizing temperature. In addition, the hot water sanitizing
rinse should have a flow pressure between 100 kPa (15 psi) to 170 kPa (25 psi), in order
to ensure an appropriate scrubbing action for the dislodgement of food particles. After
washing, all tableware items and utensils must be air dried and stored in an appropriate
location, away from any contamination risk.
29
Table 1.5. Minimum Washing And Sanitization Temperature Of Various Hot-Water
Sanitization Warewashing Machines (FDA Food Code, 2005).
Machine Type
Wash Temperature
Sanitization
Temperature
74°C (165°F)
74°C (165°F)
66°C (150°F)
82°C (180°F)
71°C (160°F)
82°C (180°F)
66°C (150°F)
82°C (180°F)
Single tank, stationary rack, single
temperature machine
Single tank, stationary rack, dual
temperature machine
Single tank, conveyer, dual
temperature machine
Multi-tank, conveyer, multi
temperature machine
1.7.2. Manual Warewashers
As specified by the 2005 FDA Food Code (4-301.12), a manual warewasher must
have at least 3 compartments for manual washing, rinsing and sanitization. Furthermore,
the sink compartments should be large enough to accommodate the immersion of the
largest equipment and/or utensils. If the sinks are not large enough, alternative manual
warewashing equipment or a 2-compartment sink can be used upon approval. This does
not apply to warewashing cleaning and sanitizing processes which have a continuous
flow of tableware items. Food establishments that reuse tableware should not use a 2compartment sink.
The washing of a tableware item is the first step in the manual cleaning procedure.
This is done with a detergent in the first compartment of the sink. The suggested
washing solution temperature should not be less than 43°C (110°F), unless specified on
the label by the detergent manufacturer. The proper concentration of detergent is also
necessary to ensure adequate removal of foodstuffs. Different foods reacts in a specific
30
way to various detergent compounds and it is imperative to understand the chemistry of a
variety of cleaning compounds and their affect on removing food stuck on tableware and
utensils (Schmidt and Rodrick, 2003).
The second compartment contains clean and potable water. This second step is to
remove all detergents and other cleaning agents from tableware items. It is accomplished
by completely immersing the utensils in the water. This water should not contain any
soap film or soap bubbles transferred from the first compartment. Cleaning agents left on
tableware items can interfere with the sanitizing action of the third compartment, and can
act as shield for any surviving pathogenic or spoilage microorganisms.
The third compartment of the manual washer contains either hot water or a
chemical sanitizer. If a food establishment decides to use hot water, the temperature must
be at least 77°C (171°F) and the tableware must be completely immersed for 30 seconds.
For compartments filled with a chemical sanitizer, the correct temperature and contact
time can be found on the sanitizer’s EPA-approved manufacturer’s label (Food Code,
2005). As is the case with mechanical washing procedures, the cleaned and sanitized
tableware must be air-dried and stored in a location where there is a minimal risk of
contamination.
1.8. The Importance Of A Cleaning And Sanitizing Program
The main objective of any sanitizing program is to remove all visible signs of
food soil from the surfaces of tableware and inactivate all bacteria that are present.
Furthermore, it is important to dry and store the cleaned and sanitized items in a sanitary
environment, free from excessive moisture. Several studies have shown that cloth used
31
to drying washed tableware could contain various bacteria, including Salmonella spp. and
Staphylococcus aureus (Scott and Bloomfield, 1990; Jiang and Doyle, 1999;
Kusumaningrum et al., 2002). These studies have found that these bacterial strains can
survive for hours or even days after initial contamination. Thus, contaminated equipment
and tableware, which could be considered clean and sanitized, can transfer pathogens to
newly prepared ready-to-eat foods (Lee et al., 2007). It is thus imperative that a proper
cleaning and sanitized program is used, especially at restaurant establishments where
several hundred meals are served on any given day.
According to the Food Code (2005) the correct procedure for cleaning and
sanitizing food contact surfaces is: (1) Clean, (2) Rinse, and (3) Sanitize. All three steps
require the use of water. According to Schmidt (2003), 95-99% of cleaning or sanitizing
solutions is composed of water. The two main functions of water are: (1) to carry the
detergent or sanitizer to the surface of the tableware or processing equipment and (2)
carry food soil or contamination from the surface (Hui et. al, 2003). It is consequently
essential that the water used for cleaning and sanitizing treatments be potable and be free
of pathogens.
Occasionally, water may contain minerals and other impurities that
considerably reduce the effectiveness of the detergent or sanitizer being used. Common
water impurities and the possible associated problems are listed in Table 1.6. In most
operations, it is economically unfeasible for the use of pure water, so the addition of
water treatment conditioners like sequestering agents (sodium tripolyphosphate,
polyelectrolytes) and chelating agents (sodium gluconate, ethylene diamine tetracetic
acid), can be used to reduce the build-up of these impurities (Hamann et al., 1990). Also,
adding selected chemical agents to the water can assist in the removal of food soil by
32
reducing the adhesion force between the food soil and the contact surface (Troller, 1993).
An example of such a chemical agent is a surfactant. A surfactant is commonly blended
into a cleaning agent, with its main function being the reduction of the surface tension of
water to allow a more intimate contact between the cleaning agent and the food soil
(Adams et al., 1989).
Table 1.6. Water Impurities And Associated Problems (Schmidt, 2003).
IMPURITY
Common impurities
Oxygen
Carbon Dioxide
Bicarbonates (Sodium, Calcium or
Magnesium)
Chlorides or Sulfates (Sodium, Calcium or
Magnesium)
Silica
Suspended Solids
Unusually High pH (above 8.5)
PROBLEM CAUSED
Corrosion
Corrosion
Scale
Scale & Corrosion
Scale
Corrosion and Deposition
Mediate Corrosion and Deposition; Alter
detergent efficiency
Mediate Corrosion and Deposition; Alter
detergent efficiency
Unusually Low pH (below 5)
Less Common Impurities
Iron
Manganese
Copper
Filming and Staining
Corrosion
Filming and Staining
The third and final step of the cleaning program is sanitization. The Association
of Official Analytical Chemists (AOAC) and the EPA (1979) define the sanitization of
food product contact surfaces as a process in which there is a reduction in the bacterial
contamination level by 99.999% (5 logs) in 30 seconds.
There are two types of
sanitization methods that are used: (1) Thermal and (2) Chemical. Thermal sanitization
33
entails the sole use of hot water or steam at a specific temperature and contact time for
the destruction of pathogens. Chemical sanitization, on the other hand, involves the use
of an EPA approved chemical product at a defined concentration and contact time
(Schmidt et al., 2003). The EPA strictly enforces the requirement that all sanitizers show
a 5 log reduction of bacterial contaminants, especially for new sanitizers that are in need
of approval. For new sanitizers, experimental data must demonstrate this requirement of
bacterial strains inoculated onto tableware that is subsequently treated with the sanitizer
at an appropriate concentration, temperature and contact time combination.
1.9. Cleaning Agents – Characteristics
Food soil is defined as any unwanted material (visible or invisible) on foodcontact surfaces (Schmidt, 2003). The main purpose of the cleaning step is to remove all
food soil from food contact surfaces. This is achieved by the cleaning agent as it lowers
the surface tension of water. This causes food soils to loosen their attachment to the
surface. This could be further loosened by mechanical action of scrubbing or high
pressure water.
Furthermore, the cleaning agent must be able to disperse the soil
particles throughout the cleaning solution after removing them from the food contact
surface. This prevents redeposition of soil particles. This could be enhanced by agitation
of the dispersed solution and/or continual use of fresh cleaning solution.
Although food soil originate primarily from the actual food(s) that comes in
contact with the surface of the equipment or tableware, it could also come from minerals
found in the cleaning water, residues from cleaning/sanitizing compounds and
microbiological biofilms. The composition of the food soil is thus very complex and the
34
appropriate cleaning must be selected in order to remove the resident type of soil. Table
1.7 shows several major types of food soils, their solubilities, removal difficulty and
behavior to heat.
Table 1.7. Characteristics of Food Soils
Surface Deposit
Solubility
Ease of Removal
Sugar
Fat
Protein
Starch
Water Soluble
Alkali soluble
Alkali soluble
Water soluble,
Alkali soluble
Water Soluble; Acid
Soluble
Acid Soluble
Easy
Difficult
Very Difficult
Easy to Moderately
Easy
Easy To Difficult
Monovalent Salts
Polyvalent Salts
Difficult
Heat-Induced
Reactions
Carmelization
Polymerization
Denaturation
Interactions with
other constituents
General Not
Significant
Interaction with
other constituents
The type of food, along with its physical characteristics (particle size, shape,
density) will determine the degree of bonding between the surface of a tableware item
and a food soil. This is referred as the adhesion forces (Handojo et. al, 2009). The
adhesion strength of the soil to the contact surface is also directly related to the
environmental humidity and time of contact. Table 1.7 shows that protein and fat-based
food soils are very difficult to remove.
Usually, these food soils are found in
combination and removing them may require dual purpose cleaning agents.
It is
generally known that acidic cleaning agents dissolve soils that are alkaline (minerals),
while basic cleaners dissolve acidic-based food soils (Mariott and Gravani, 2006).
Improperly using detergents can actually make the food soil more difficult to remove
35
soils. Also, cleaning immediately after soiling the equipment or tableware is much easier
than letting the food soil dry, as a “baked-on” deposit. It is thus important to identify the
difficulty required to remove various food soils from contact surfaces. A new analytical
method that is gaining interest is the use of Atomic Force Microscopy.
1.9.1. Atomic Force Microscopy
Atomic force microscopy (AFM) is a technique used to measure and produce a
three-dimensional image of extremely small particles, in the nano-scale (Binnig et al.,
1987). This method has been used to image metal surfaces and has recently been used to
image soft samples, including biological (proteins, DNA, whole cells) and food samples
(Radmacher et. al, 1992; Henderson, 1994; Handojo et al., 2009). The AFM process,
displayed in Figure 1.5, has a sharp tip that is attached to a cantilever spring, and which is
brought in close proximity of the surface of a sample during a test (Verran et al., 2000).
During scanning, the forces between the surface and the tip cause deflections in the
cantilever. Concurrently, a laser beam is focused on the surface of the cantilever, and the
deflections are relayed by the laser beam to a photodiode and then sent to a detector
which ultimately creates a topical three-dimensional image of the surface (Allen et al.,
1997; Smith, 1999). AFM is therefore different from traditional imaging instruments
because is measures the attractive and repulsive forces between the tip and the surface of
a sample rather than physically looking at the surface (Morris, 2004).
Generally, as two different materials (the tip and the sample) approach each other,
atoms from both surfaces develop attractions due to Van der Waals forces. As the tip and
sample surface continue to approach each other, the atoms are electrostatically repelled,
36
thus causing deflections in the cantilever. Other tip-surface sample forces that may cause
deflections are capillary forces, magnetic forces and fluid surface tension (Cleveland et
al., 1998). There are three common AFM operating modes: (1) non-contact, (2) contact
and (3) tapping.
Figure 1.1. Diagram of AFM Imgaing
Process (Absolute Astronomy, 2009).
Figure 1.2. Image Of A Cantilever And
Tip (Wiesner Research Group, 2009).
In the non-contact AFM mode, the cantilever oscillates slightly above the surface
(no direct contact) at a resonant frequency (Binnig et al., 1987). In this mode the tip
never touches the sample, but, due to the attractive and repulsive forces between the tip
and the surface, the amplitude of the cantilever changes to reflect the underlining
topography. This particular AFM mode minimizes degradation of the sample surface,
especially during the analysis of food samples (Kelsall et al., 2005).
37
The contact AFM mode brings the tip in constant physical contact with the
surface of the sample. In this mode, the tip is dragged along the test area, and the
cantilever deflections that correspond to the surface are measured and converted into a
topographical image. This AFM mode has gained interest in the food industry, especially
when trying to remove a section of a samples surface with a specified force (Bhushan and
Koinkar, 1994). This procedure can be useful when trying to determine the thickness of a
film that is attached to a surface. An example of this can be seen in a study done by
Handojo et al. (2009). During Handojo’s study, the AFM contact mode was used to
scratch a specified area of a milk film attached to a glass surface. A cross-sectional view
of the scratched area was then used to measure the thickness of this film.
The tapping mode is a hybrid form of the non-contact and contact modes (Putman
et al., 1994). In the tapping mode, the cantilever oscillates vertically and during each
oscillation cycle, the tip briefly makes contact with the surface of the sample at a constant
amplitude (Raghavan et al., 2001). As the tip comes in contact with the sample’s surface,
changes occur in the tip’s oscillation parameters. These changes are then used to create a
topical 3-D image of the surface. This mode is advantageous due to the fact that the
image is of higher quality and with better resolution, especially when testing “soft”
samples like food deposits on surfaces. As stated by Putman et al. (1994), due to the high
oscillating frequency of the cantilever, soft biological samples behave as “hard” materials.
As a result, the surface is less susceptible to deformations than when using the contact
mode.
38
1.10. Classes of Cleaning Detergents
Types of cleaning agents that are commonly used in processing facilities and food
service establishments are (1) soaps, (2) alkaline-based, (3) acid-based, and (4) synthetic
detergents. As stated by the FDA (1995), these cleaning agents must be safe on contact
by employees, non-corrosive to the food contact surface, easily and completely dissolved
and stable under normal storage conditions. These cleaning agents are sometimes mixed
with other chemical compounds that are specifically formulated to remove certain soils or
to remove difficult strongly bonded mineral deposits.
1.10.1. Soaps
One of the best-known and oldest cleaning agents is plain soap. Soaps are
generally best for the removal of oils, fats and greases.
This is achieved by the
suspension of the fat particles in the soap solution, in a process called emulsification.
This process is the physical breakdown of fats and oils into smaller globular particles that
are subsequently dispersed throughout the soap medium (Hui et al., 2003). As displayed
in Figure 1.3, the soap compound has a hydrophilic end which binds to the water in the
soap solution, while the hydrophobic (fat and oil soluble) end binds to the soil. As a
result, soap molecules are able to surround soil particles and form micelles (Figure 1.4).
These micelle droplets are then miscible in water and can be washed away easily. It is
important to note that soap is not generally used in processing plants and food service
establishments due to its limited cleaning functionality and the fact that it may react with
hard water to form insoluble curds.
39
Figure 1.3. Anionic Surfactant Molecule (Mariott
and Gravani, 2006).
Figure 1.4. Soil Particle
Suspended By Micelle Formation
(Mariott and Gravani, 2006).
1.10.2. Alkaline Cleaning Agents
Alkaline cleaning compounds are generally best used for the removal of fats, oils
and proteins. Alkaline, by definition is any compound with a pH greater than 7 (Fellows,
2005). As the pH increases, the alkalinity of the compound increases as well. This type
of cleaning agent is categorized as: (1) strongly alkaline, (2) heavy-duty alkaline and (3)
mild alkaline cleaners. The first two alkaline subclasses are known to have very strong
dissolving and cleaning actions, but are highly corrosive to aluminum, tin, galvanized
metal, glass and hazardous to humans. These types of cleaning compounds are generally
used for tough cleaning jobs, mainly in processing plants, especially for Clean-In-Place
(CIP) systems (Schmidt, 2003). An example of a strongly alkaline cleaning agent is
sodium hydroxide (NaOH) or also known as caustic soda (McSwane et. al, 2005).
Caustic soda is known to cause burns on the skin and inhalation of the fumes may cause
40
respiratory damage. The third class of alkaline cleaners are the mild ones such as sodium
bicarbonate, sodium sequicarbonate and sodium metasilicate (Skaarup, 1995). This class
is much less corrosive than the first two classes and is usually used for manual cleaning
of lightly soiled surfaces, especially in the food service industry.
1.10.3. Acidic Cleaning Agents
Acidic cleaning agents are not as commonly used in the food industry as are
alkaline-based compounds. This is due to the fact that acid cleaners are not as effective
in removing fats, oils and proteins. Acidic compounds are considered specialized types
of cleaners and are specifically used to dissolve mineral scale deposits (calcium and
magnesium precipitates) and to remove encrusted food soil and hard water deposits from
food-contact surfaces (Mariott and Gravani, 2006). Typically, acid detergents are used in
a two step cleaning process in conjunction with alkaline cleaners. Acidic cleaners can be
classified as being either an inorganic acid or an organic acid (Schmidt, 2003). Inorganic
acids are proficient in removing mineral deposits, but they can be particularly irritating to
human skin and may be corrosive to surfaces (metals, concrete, and fabrics). These
inorganic acids (hydrochloric, sulfamic, hydrofluoric) are generally used in processing
facilities that have boilers and steam-producing equipment. Organic acids (tartaric acid,
citric acid), on the other hand, are not corrosive or irritating to skin. They are excellent
water softeners and are commonly used in manual cleaning procedures at food-service
establishments. The downside of organic cleaners is that they are much more expensive
than inorganic cleaners.
41
1.10.5. Synthetic Detergents
Synthetic detergents are essentially a soap cleaner, but one that will not form
curds when exposed to hard water (high in impurities). These synthetic detergents are
formed when different cleaning components are mixed together to provide the same
emulsification function of soap on fats, greases and oils. Another advantage of synthetic
cleaners is that they contain surfactants which are intended to lower the surface tension of
the cleaning solution. By lowering the surface tension, there will be higher “wetting” of
the soil particles occurs, and this increases the detachment of the soil from the contact
surface (FDA, 1995). The advantages that surfactants add to synthetic detergents are
emulsification, dispersion, noncorrosiveness, nonirritation and they can be rinsed easily
from surfaces.
1.11. Sanitizing Agents – Characteristics
After the cleaning and rinsing steps, the final step functions to inactivate any
pathogenic microorganisms of concern which may still be present on tableware or
processing equipment. The definition of “sanitization”, as described in the FDA Food
Code (2005), is the application of cumulative heat or chemicals on cleaned food-contact
surfaces that, when evaluated for efficacy, is sufficient to yield a reduction of 5 logs,
which is equal to a 99.999% reduction, of representative disease microorganisms of
public health importance. Chemical sanitizers must show this efficacy before being
approved to the EPA (1979). Furthermore, sanitizers used in the food industry must be of
approved food-grade quality since any chemical residue that enters the food supply must
be deemed safe (FDA Code of Federal Regulations, 21 CFR 178.1010).
42
Table 1.8 shows several physical and chemical factors that are known to affect the
efficacy of sanitizers. These factors are usually interconnected, meaning that one or more
factors can be adjusted to compensate for a factor that is inadequate. For example, if a
certain sanitizer requires preparation in a cold water solution, increasing exposure time or
sanitizer concentration can compensate for the low application temperature.
Table 1.8. Physical And Chemical Factors Affecting Sanitizer Effectiveness (Schmidt,
2003; Mariott and Gravani, 2006; Hui et. al, 2003).
Factors
Surface cleanliness
Exposure Time
Temperature
Concentration
Water Hardness
Microbial Population
Advantages & Disadvantages
An unclean surface cannot be sanitized because chemical
sanitizers lack penetration ability and requires direct contact
with microorganisms.
Cracks, pitts, and crevices on food-contact surfaces should be
avoided.
Generally, the longer contact time, the more effective the
sanitization effect.
An increase in temperature generally increases microbial
inactivation. However, chemicals are more corrosive at higher
temperatures. Thus, chemical sanitizers should be applied at
ambient temperatures.
An increased concentration increases sanitizer efficacy, to a
certain point. There is a specific maximum concentration,
where efficacy does not increase by increasing concentration.
As water hardness increases, the effectiveness of sanitizers
decreases.
The higher the initial microbial load, the higher the possibility
of survivors after the sanitization step.
43
An ideal sanitizer should have the following properties:
•
•
•
•
•
•
•
Broad-spectrum biocidal activity against bacteria, yeasts, molds and
viruses
Rapid inhibition of pathogenic microorganisms
Stable and effective under a broad range of environmental conditions
(presence of organic soils, water hardness, pH)
Low toxicity and corrosivity
Ease of use
Readily available
Inexpensive
There are numerous chemical agents that are effective in inactivating pathogenic
microorganisms, but unfortunately they often have disadvantages that make them less
than ideal. Some sanitizers may stain surfaces, may be highly corrosive, leave films on
the surface and may impart an off-flavor to freshly prepared foods. Other chemicals may
be too costly or may be extremely hazardous to employees.
1.11.1. Chlorine
Chlorine sanitizers are one of the most commonly used chemicals in the
processing of fresh produce, as well as the sanitization of equipment and utensils
(Fabrizio et al., 2002; Tasi et al., 1992). Chlorine is available in a variety of forms,
including hypochlorite, liquid chlorine, organic and inorganic chloramines (Kreske et al.,
2006). The basic mode of biocidal action is primarily on the cellular membranes of
microorganisms by inhibiting the enzymes involved in glucose metabolism (Wei et al.,
1985; Banwart, 1989). Other mechanisms of inactivation that has been proposed are: (1)
disrupts protein synthesis, (2) inhibits the uptake of oxygen, (3) causes the leakage of
macromolecules and (4) negatively affects the cells DNA properties (Dychdala, 2001).
44
It is important to note that microorganisms are only affected by “free available
chlorine” (FAC) and not by bound chlorine. There are two forms of chlorine that are
considered FAC: (1) hypochlorite ion (OCl-) and (2) hypochlorous acid (HOCl). It has
been concluded by many research publications that the hypochlorous acid is the most
active form, being 80 times more effective in sanitization applications than the
hypochlorite ion form, at the same concentration (Cords and Dychdala, 1993; Wei et. al,
1985). The amount of HOCl that is formed in solution is a function of the pH of solution.
A lower pH level fosters HOCl formation, with a solution of pH 5 having nearly all
chlorine in the HOCl form (Bloomfield and Arthur, 1994). A higher pH increases the
formation of OCl- ions, and thus decreases the efficacy of chlorine-based sanitizers.
When the pH is at 4.0 or lower, deadly Cl2 gas (mustard gas) can form, and this could be
a safety and health concern due to irritations to the skin and damage to mucous
membranes (Hinton et al., 2007).
In addition to pH, the efficacy of chlorine chemicals are also affected by the
temperature of the solution and the presence of organic food soils. The chlorine solution
that is used for sanitization of food-contact equipment and tableware should be at a
minimum temperature.
This is based on the pH and concentration of the chlorine
solution as displayed in Table 1.9. Furthermore, chlorinated chemicals should not be
applied at higher temperatures since this increases its corrosive properties. The presence
of organic soils also negatively impacts the effectiveness of chlorine compounds since
they reduce the amount of FAC in solution (McSwane et al., 2005; Wei et al., 1985). The
advantages and disadvantages of chlorine-based sanitizers are summarized in Table 1.10.
45
Table 1.9. The Minimum Temperature Of Chlorine Sanitizing Solutions For FoodContact Surfaces At A Certain pH And Concentration (FDA Food Code, 2005).
Minimum Concentration
Mg/L (ppm)
Minimum Temperature
25
pH 10 or less
C° (F°)
49 (120)
pH 8 or less
C° (F°)
49 (120)
50
38 (100)
24 (75)
100
13 (55)
13 (55)
Table 1.10. The Advantages And Disadvantages Of Chlorine-Based Sanitizers (Schmidt,
2003, Mariott and Gravani, 2006; Wei et. al, 1985; Beuchat et al., 2004).
Advantages
•
•
•
•
Disadvantages
Effective against a variety of
bacteria, fungi and viruses
Inexpensive
Unaffected by water impurities
Leaves minimal residue or film on
surfaces
•
•
•
•
•
•
46
Corrosive to stainless steel and
other metals
At pH 4.0 or lower, toxic and
corrosive gas is formed.
Higher
temperatures
promote
corrosive action
Skin irritant and damage to mucous
membranes.
Negatively affected by the presence
of organic material
Possible formation of carcinogenic
trihalmethanes (THM) compounds
under certain conditions.
1.11.2. Quaternary Ammonium Compounds (QAC’s)
Quaternary ammonium sanitizers are approved for use in the United States on
tableware, food processing equipment, and other food contact surfaces, with a maximum
concentration of 200 parts-per-million (ppm) (FDA, 2003).
Quaternary ammonium
compounds are a class of compounds with a structure, displayed in Figure 1.3. QAC’s
are synthetically created by a nucleophilic substitution reaction between a quarternizing
agent and a tertiary amine and produces a nitrogen atom covalently linked to four organic
alkyl groups (Mariott and Gravani, 2006). This a positively charged ion that is ionically
bonded to an anion, usually chlorine. The properties of QAC’s depends greatly upon the
alkyl groups (R1, R2, R3 and R4 groups), which can vary tremendously.
Figure 1.5. Chemical Structure of QAC (Schmidt, 2003).
Since QAC’s are positively charged, the primary mode of inhibitory action
against microorganisms is their attraction to the bacterial proteins and cell membranes,
which are negatively charged (Trauth et. al, 2001). This association disrupts proteins and
the extracellular functions, thus inhibiting the pathogenic and spoilage action of the
organism (McDonnell and Denver, 1999). According to the FDA Food Code (2005), in
order for QAC’s to be effective, the water hardness should be 500 mg/L or less, at a
47
minimum temperature of 24°C (75°F) and be at a concentration that is listed on the
manufacturers label.
QAC’s are colorless, odorless, generally nontoxic and non-corrosive.
Other
advantages include its better stability in the presence of organic food soils when
compared with chlorinated compounds. QAC’s are also stable over a broad pH range and
at high temperatures. However, although QAC’s are effective against Gram-positive
bacteria, Gram-negative bacteria are not extensively affected (Trauth et. al, 2001).
QAC’s are also incompatible with negatively charged synthetic detergents and soaps.
Thus for maximum effectiveness, it is essential to thoroughly rinse the contact surfaces
from residual detergents before using QAC.
The advantages and disadvantages of
QAC’s are summarized in Table 1.11.
Table 1.11. The Advantages And Disadvantages of QAC’s (Schaeufele, 1984).
•
•
•
•
•
•
Advantages
Colorless and odorless
Non-irritating to skin, non-toxic
Non-corrosive
Stable over a broad range of pH and
temperatures.
Provides a residual antimicrobial
film
More stable in the presence of
organic materials than chlorine
compounds
48
•
•
•
Disadvantages
Inefficient with Gram-negative
microorganisms
Incompatible with soaps and
anionic synthetic detergents
Residual films are not advantageous
to cultured dairy products, cheese
and beer food processing operations
1.11.3. Acidic-Based Sanitizers
Acidic-based sanitizers are similar in action to QAC’s by their surface-active
characteristics (Schmidt, 2003). Usually, these sanitizers are mixed with a surfactant and
are used to combine the rinsing and sanitizing steps. Examples of various acidic-based
sanitizers include acetic acid, peroxyacetic, lactic and propionic acids.
Unlike QAC’s, acid sanitizers are negatively charged and consequently has the
ability to neutralize any residual alkaline residue from cleaning detergents (Hui et. al,
2003). This neutralization prevents the formation of alkaline deposits on the surface.
This can also be a disadvantage if too much residual detergent is left on the surface. This
is so because more active ingredients will be used to neutralize the detergent and less will
be available for inactivation of pathogens of concern. Additionally, acidic sanitizers are
capable of effectively inactivating microorganisms of public health concern. The mode
of this inhibitory action is the attraction of the negatively acidic surfactants to the
positively-charged cell membranes of the bacteria (Banwart, 1989).
This attraction
allows for the penetration of acid compounds into the interior of the cell and as a result
the acidification of the cellular cytoplasm (Fabrizio et. al, 2002). Additional advantages
and disadvantages are shown in Table 1.12.
49
Table 1.12. The Advantages And Disadvantages Of Acidic-Based Compounds.
Advantages
•
•
•
•
•
Disadvantages
Includes a surfactant – Good
wetting properties
Non-toxic
and
Non-corrosive
(permits exposure to equipment and
tableware for overnight soaking)
Biodegradable
Low odor-potential
Very stable
•
•
•
•
Relatively high cost
Narrow pH range (pH 2-3)
Low activity on yeasts and molds
If too much alkaline detergent
residue is left on the surface, acidic
sanitizers will be neutralized and
little sanitization can occur.
1.11.4. Neutral Electrolyzed-Oxidizing Water (NEW)
Compared with the other sanitizers discussed earlier, electrolyzed-oxidizing (EO)
water is relatively new. It has been used for several years in Japan, as a disinfectant for
the hands and as a cleaning agent for beds and floors in medical institutions (Horiba et al.,
1999; Fabrizio et. al, 2002). Recently, EO water has gained interest as a sanitizer in
agriculture, dentistry, medicine and the food industries and is currently approved for use
in the United States by the EPA (Park et al., 2002; Huang et. al, 2008). Numerous
publications have stated that EO water has powerful biocidal action against
microorganisms of concern (Listeria monocytogenes, Escherichia coli O157:H7,
Salmonella Enteritidis, Campylobacter jejuni, Bacillus cereus). It is also effective in
reducing foodborne pathogens on cutting boards and various food products such as
poultry and fresh produce, and effective in eliminating L. monocytogenes biofilms on
stainless steel (Horiba et al., 1999; Venkitanarayanan et al., 1999; Kim et. al, 2001; Park
et al., 2002; Deza et. al, 2005).
50
EO water is created by passing a dilute salt (NaCl) solution though a generator
containing a cell that contains a positively charged anode and a negatively charged
cathode, separated by a permeable membrane (Figure 1.5) (Huang et al., 2008). These
electrodes are subjected to a direct current and consequently, negatively-charged ions
(chloride and hydroxide) from the NaCl solution move to the positive anode and give up
electrons to produce oxygen, chlorine gas, hypochlorite ion, and hypochlorous acid. At
the same time, positively-charged ions (hydrogen, sodium) move to the cathode to take
up electrons and become sodium hydroxide and hydrogen gas (Hsu, 2005). As a result of
this procedure, two types of water are generated concurrently. The water that is directed
to the cathode produces electrolyzed reduced water, which has a pH of 11.4 and an
oxidizing-reduction potential (EH) of -795 mV (Kim et al., 2000). The water that is
produced at the anode side is the EO water that is used as a disinfectant for various
washing and sanitizing procedures. EO water is characterized as being acidic when it has
a pH of less than 2.7, an Eh of +1000 mV and the presence of hypochlorous acid. (Horiba
et al., 1999). More recently, the food industry has become interested in the use of neutral
EO water (NEW) since its pH is approximately 6.5 and it is less corrosive to food-contact
surfaces. NEW is produced by redirecting the products formed at the cathode towards
the anode chamber. This gives rise to a solution characterized with a pH in the range of
5.5-7.0 and an Eh of 600-800 mV (Horiba et al., 1999).
51
Figure 1.6. The Schematic Process Of Electrolyzed Water Generator (Huang et al., 2008).
The inactivation of microorganisms of concern by NEW is due to the high Eh and
the presence of hypochlorous acid. Eh is defined as the ability to accept or lose electrons
(Jay et. al, 2005). A highly positive Eh value, a characteristic that NEW possesses,
indicates the acceptance of electrons. Accordingly, NEW sequesters electrons from the
cellular membrane of microorganisms, allowing for the hypochlorous acid to move into
the cytoplasm of the cell and eventually inhibits its functions. This is similar to the
biocidal action from chlorine-based sanitizers (Fabrizio et al., 2002). At the same time,
the HOCl generated with the EO water is much more environmentally friendly and less
toxic to humans when compared with the HOCl compounds found in chlorinated
solutions. This is due to the stabilizing combination of elemental chlorine (Cl2) and
caustic soda (NaOH) that is required to create sodium hypochlorite (Mariott and Gravani,
52
2006).
Further evidence of the inhibitory action of EO water is evidenced from
experiments performed by Osafune et al., 2006. In that study, they used a scanning
electron microscope to view Staphylococcus saprophyticus, Micrococcus luteus and
Bacillus sphaericus after being treated with EO water. The results showed that cells
exposed to EO water wrinkled walls with round pores from which the cytoplasmic
materials had leached.
Although EO water is a relatively new sanitizer, available published data have
reported various advantages that far outweigh its disadvantages, when compared with
traditional sanitizers like chlorine and quaternary ammonium. One of the most important
benefits of using EO water is its safety to humans and the environment due to the fact
that EO water is generated from tap-water and sodium chloride (Kim et al., 2000).
Furthermore, EO water is usually produced on-site and immediately before use, it
eliminates the need for the handling of concentrated toxic chemicals (Park et al., 2002).
NEW is also non corrosive to the skin and mucous membranes and is more stable when
compared with acidic EO water (Hiratsuka et al., 1996; Horiba et al., 1999). After the
initial capital investment resulting purchase of an EO water generator, the only operating
expenses are water, NaCl, electricity and its maintenance (Walker et al., 2005).
1.11.5 Summary Of The Sanitizers Previously Discussed
Sodium hypochlorite and QAC are traditional sanitizers that are commonly used
in the food industry. While they are effective in inactivating pathogens of public health
concern, they may be corrosive to equipment, hazardous to humans and may have a
negative impact on the environment. As a result, the need to develop alternative sanitizers
53
is urgent. Examples of these alternative options are organic acid-based sanitizers and
electrolyzed-oxidizing water. These are less hazardous to humans, less corrosive and are
more environmentally friendly.
Table 1.13. is a summary of the advantages and
disadvantages of the four sanitizers previously discussed.
Table 1.13. Summary Of The Advantages And Disadvantages Of Chlorine, QAC,
Acidic-Based And EO Water Sanitizers
Advantages
Disadvantages
Chlorine
• Effective against a variety of
pathogens of concern
• Inexpensive
• Unaffected by water impurities
• Corrosive
• Skin irritant and damage to
mucous membranes
• Negatively affected by the
presence of organic material
• Possible formation of carcinogenic
trihalomethanes (THM)
compounds under certain
conditions
QAC
• Non-corrosive, non-irritating,
non-toxic
• Residual antimicrobial film
• More stable in presence of
organic materials than chlorine
• Incompatible with soaps
• Ineffective in hard water
• Residual films are not
advantageous to cultured dairy
products
Acidic
• Includes surfactant
• Non-toxic, non-corrosive
• Biodegradable
• Very stable
• Non-toxic, Non-corrosive
• Environmentally friendly
• Biodegradable
• High Eh value – inactivation of a
variety of pathogens of concern
• Eliminates storage of highly
concentrated chemicals
• Relatively high cost
• Low activity on yeasts and molds
• Narrow pH range (pH 2-3)
• Reacts with alkaline detergents
• High initial capital investment
• Maintenance and repair costs
EO Water
54
CHAPTER 2
COMPARATIVE EFFICACIES OF VARIOUS CHEMICAL SANITIZERS FOR
WAREWASHING OPERATIONS IN RESTAURANTS
2.1. Abstract
Recent reports have stated that contamination of food contact surfaces is a major
problem in restaurants. As a result, cleaning and sanitization of all utensils should be
maximized since this minimizes the risk of cross-contamination. The FDA Food Code
(2005) states that 5 log/CFU bacterial reductions must be obtained during tableware
cleaning protocols. However, there is little published data on the comparative efficacies
of various sanitizers for the accomplishment of this goal. Our study investigated the
survival of Escherichia coli K12 and Listeria innocua inoculated into cream cheese and
pasteurized whole milk and subsequently used to soil ceramic plates, plastic serving trays
and drinking glasses. These items were washed with automatic 49°C (120°F) and manual
43°C (110°F) dishwashers, then treated with electrolyzed-oxidizing water (EO),
quaternary ammonium compound, acidic and hypochlorite sanitizers. For the lowest
efficacies, the results showed that after six and eight washing cycles in the same solution,
≥5 log reductions were achieved in the manually washed E. coli K12 contaminated trays
55
and plates, respectively, which were exposed to the chlorine and quaternary ammonium
sanitizers. For Listeria, seven and eight washes produced the same results for trays and
plates, respectively. For automatic washing, the minimum wash cycles for: (1) E. coli
K12 were seven and nine for the trays and plates, respectively using the EO and chlorine
sanitizers and (2) L. innocua were seven and ten for the trays and plates, respectively
using the ammonium and chlorine sanitizers. Drinking glasses contaminated with: (1) E.
Coli K12 showed that 14 and 16 washing cycles achieved ≥5 log reduction for manual
and automatic washing, respectively, and (2) L. innocua showed a maximum of 14 cycles
for manual washing and 17 cycles for automatic washing before the inability to produce
the 5-log bacterial reduction. Information obtained from this study could be used by
restaurants and other foodservice institutions to minimize the cost of food contact surface
cleaning while still meeting FDA mandates.
2.2. Introduction
Foodborne illness continues to be a public health concern in the United States,
especially in the food-service industry. The National Restaurant Association (2009)
reports that the year 2009 will show sales of $566 billion in prepared foods. On a typical
day, more than 130 million individuals will eat at a foodservice establishment. Thus, it is
vital that not only should establishments provide a safe and high-quality meal, they
should also reduce the factors that contribute to foodborne illnesses. Three of the most
important factors that contribute to foodborne illnesses are: (1) incorrect temperatures
56
during storage/processing, (2) poor personal hygiene of workers and (3) crosscontamination (Wernersson et al., 2004).
The first factor is considered the major contributor to foodborne illnesses, while
the other two factors are believed to play a role in foodborne illness but to a lesser degree
(Cogan et al., 2002).
This factors are due to a high turnover of the workforce in
restaurants since most employees are young and consider this a temporary occupation. It
is thus very difficult and costly to educate individuals about good personal hygiene and
proper techniques that minimize cross-contamination.
The Food Code (2005) and NSF International (ANSI/NSF 3, 2005) require the
complete removal of food soil from contact surfaces after the completion of washing and
sanitization protocols. If food soils that are contaminated with microorganisms are not
properly removed during the washing step, bacteria may survive the sanitation step due to
the fact that the cells may be shielded by the food (Wernersson et al., 2004).
Additionally, the sanitization step requires that the application of heat or chemicals on the
food contact surface must yield a 5-log (99.999%) reduction in representative disease
microorganisms. Chemical sanitizers that are available for use must meet this mandate of
efficacy. Traditional sanitizers include paracetic acid, iodophors, ultraviolet radiation,
ozone, sodium hypochlorite and quaternary ammonium compounds (QAC) (Gavin and
Weddig, 1995). These sanitizers, when used at the approved concentration, temperature
and contact time have shown an adequate ability to reduce microbial loads to acceptable
levels. However, despite this acceptable bacterial reduction, there are drawbacks with the
use of these traditional sanitizers. These chemicals can be highly corrosive to equipment,
57
toxic to the environment and harmful to workers and consumers (Ruiz-Cruz et al., 2007;
Ukuku, 2006). As a result, there is a need to develop new sanitizers with the ability to
produce the mandatory microbial reduction, while being more environmentally friendly,
less corrosive to equipment and less toxic to humans. Two of these sanitizers are neutral
electrolyzed-oxidizing water (NEW) and PRO-SAN®. PRO-SAN® is an organic acidic
sanitizer made from natural ingredients, while NEW is generated by salt (NaCl), tap
water and an electrolyzed-oxidizing unit.
During their normal operations, restaurants and food-service establishments
usually wash several batches of tableware items in the same lot of cleaning agents. This
is done as a cost saving method. However, there is a need to determine the maximum
number of warewashing cycles that can still achieve a 5-log bacterial reduction as
required by the Food Code. This study focused on comparing the efficacy of two
traditional sanitizers and compared them to two novel sanitizers. This was done by
comparing the maximum number of warewashing cycles that a single lot of cleaning
agents can handle and still produce a 5-log bacterial reduction.
The choice of materials and parameters used in this study were carefully chosen
so that a “worst-case scenario” was created. As a result, the choice of material type used
for the utensils were ceramic plates, drinking glasses and plastic trays. The choice of
food types used to contaminate the utensils were cream cheese (non-water soluble) and
whole milk (water soluble). The bacteria selected to inoculate the food samples were
Gram-positive and Gram-negative species. In order to simulate foodservice operations,
both manual and mechanical warewashing protocols were tested.
58
The objectives of this study were:
™ To compare the cost-effectiveness of sodium hypochlorite, QAC, NEW and PROSAN® as sanitizers for tableware washing protocols.
™ To compare the efficiency of three-compartment manual warewashing with spraytype mechanical warewashing operations.
™ To evaluate the effect of material-type (ceramic vs. plastic vs. glass) on the
efficiency of the washing and sanitizing protocols.
™ To investigate the survivability of Gram-positive Listeria innocua and Gramnegative Escherichia coli K12 on tableware items after washing protocols.
2.3. Materials and Methods
2.3.1. Preparation of Bacterial Cultures
Escherichia coli K12 (ATCC 29181) and Listeria innocua (ATCC 33090) were
used in this study. Stock cultures of E. coli K12 and L. innocua were stored in a -80°C
freezer in 30% (v/v) sterile glycerol (Fisher Scientific, Fair Lawn, NJ) and the bacterial
cells were revived when required for the experimental procedure.
When needed for testing, the frozen E. coli and L. innocua cultures were allowed
to temper to room temperature and then a loopful was transferred into each of two
respective bottles containing 50 mL Trypticase soy broth (Difco Laboratories, Sparks,
MD) containing 0.3% (w/w) yeast extract (Fisher Scientific, Fair Lawn, NJ) (TSBYE),
followed by incubation at 35-37°C for 18 h. After this incubation, a loopful of the broth
was then spread on a Trypticase soy agar (Difco Laboratories, Sparks, MD)
59
supplemented with 0.3% (w/w) yeast extract (TSAYE) slant and incubated at 35-37°C for
24 h. This TSAYE slant was subsequently stored in a refrigerator at 3°C and used as a
stock culture.
Preceding each experimental run, a loopful of either E. coli K12 or L. innocua
stock culture was propagated aerobically in 200 mL TSBYE at 35-37°C for 18 h.
Subsequently, a 185 mL aliquot of each cell broth was centrifuged (Kendro Laboratory
Products, Sorvall RC 5C Plus, Newtown, CT) at 6,000 RPM for 10 min at 4°C to suspend
the cells into a pellet.
The supernatant was decanted and the cell suspension was
resuspended in 185 mL of sterile deionized potassium phosphate buffer (pH 7.2) until an
initial concentration of approximately 1.0 x 109 CFU/ml for both E. coli K12 and L.
innocua were achieved. The buffer stock solution was prepared by mixing 0.1 M
potassium phosphate dibasic (Fisher Scientific, Fair Lawn, NJ) and 0.1 M potassium
phosphate monobasic (Acros, New Jersey, NJ) to reach a neutral pH of 7.2. The bacterial
population of each inoculum was enumerated after pipetting 1 mL of appropriate
dilutions on duplicate TSAYE plates and incubating them at 35-37oC for 36 h. Each
bacterial cell suspension was then thoroughly mixed into the given food sample.
2.3.2. Preparation of Food Samples
Pasteurized whole milk and soft cream cheese were the food samples that were
used to soil the surface of the selected tableware. The food samples were purchased from
a local grocery store (Columbus, OH) one day prior to each experimental run and stored
in an incubator at 4 ± 1°C. Prior to inoculation, a 1440 mL aliquot of milk or a 1500 g
60
portion of soft cream cheese was weighed into a sterile beaker. The bacterial cell
suspension of either E. coli or L. innocua, 1:10 w/w, was then added to the beaker
containing either semi-solid cream cheese or whole milk and blended for 10 min to
ensure adequate mixing of the inoculum and food sample.
Three different tableware items were used in this study: (1) plastic serving trays,
(2) ceramic plates and (3) drinking glasses. All tableware items were thoroughly cleaned
and hot water sanitized before each experimental run.
Samplings of these items
confirmed that they were bacteria free after this cleaning step. Based on the method
selected by Lee et al. (2007), 5 g of contaminated cream cheese was applied to the entire
food-contact surface of each ceramic plate, while 10 g of cream cheese was spread on the
entire food-contact surface of each plastic serving tray. The inner wall of each drinking
glass, on the other hand, was contaminated with 0.5 g of inoculated whole milk. The
tableware items were air-dried for 1 hour at 24 ± 2°C to simulate the common procedure
that occurs at restaurants and food-service establishments. To determine if there was an
effect of the air drying on the viability of the bacterial colonies, the tableware items were
sampled using a sterilized cotton swab before and after the air-drying procedure. This
swab was subsequently serially diluted and bacterial colonies were enumerated on
TSAYE. As displayed in Figure 2.1, 16 plates was considered one warewashing cycle,
while 8 trays and 25 drinking glasses were considered one warewashing cycle. One
experimental run consisted of the use one type of tableware and one type of chemical
sanitizer and was repeated three times (Figure 2.2).
61
A
B
C
Figure 2.1. Tableware Used In Experimental Procedure (A) Plastic Serving Trays, (B)
Ceramic Plates and (C) Drinking Glasses.
2.3.3. Preparation Of Detergent And Sanitizer Solutions
Detergents
Two detergents were used in the washing step: (1) Guardian Score detergent
(Ecolab, Inc., St. Paul, MN) for the mechanical washer and (2) Mag Fusion detergent
(Ecolab, Inc., St. Paul, MN) for the manual warewashing operations. The Guardian
Score detergent was used at 1,000 ppm and its ingredients included sodium hydroxide,
sodium dichloroisocyanurate, sodium carbonate, sodium chloride, and sodium
tripolyphosphate. The Mag Fusion detergent was used at 100 ppm concentration and was
composed of sodium dodecylbenzene sulfonate, acetic acid, sodium salt, poly(oxy-1,2ethanediyl), alpha.-sulfo-.omega.-hydroxy-, C10-16-alkyl, ethers, amides, coco, n(hydroxyethyl), d-glucopyranose, oligomeric, C10-16-alkyl glycosides and fatty alcohol
alkanolamides.
These concentrations were recommended by the respective
manufacturers.
62
Manual Dishwashing
Mechanical Dishwashing
Listeria innocua
Escherichia coli K12
EO Water
QAC
Chlorine
PRO-SAN
63
Plate
(cream cheese
contaminant)
Serving Tray
(cream cheese
contaminant)
Bacterial count before and after cleaning
Figure 2.2. Experimental Design Of Warewashing Procedure.
Glass
(whole milk
contaminant)
Sanitizers
Four sanitizers were used in this study: (1) Electrolyzed-oxidizing water, (2)
PRO-SAN® (acidic-based sanitizer), (3) Quaternary Ammonium Compounds (QAC), and
(4) Sodium Hypochlorite.
(1) Electrolyzed-oxidizing water
An electrolyzed-oxidizing (EO) water generator (Ecaflo 110, Trustwater Inc.,
Ireland), as shown in Figure 2.3, was used to produce the EO water. This was produced
from a saturated sodium chloride (NaCl) (Morton International Inc., Chicago, IL)
solution and a continuous flow of tap water that were fed to the EO generator (the tap
water flow rate was set at 20 gallon per hour). From these items, two water solutions are
generated: (1) catholyte and (2) analyte. The catholyte solution was produced at the
cathode side of the generator and was characterized by having a highly basic pH (~ 1112). The analyte water solution had an acidic pH (~ 2-3). However, in this study, neutral
electrolyzed water (NEW) was used as the sanitizer. In order to produce this NEW, the
generator redirected some of the catholyte solution to the analyte solution produced at the
anode. The current and voltage was set at 3.0-3.3 A and 18.5-20.5 V, respectively. This
produced a NEW solution output of 1.0 L/min.
64
Figure 2.3. EO Water Generator Used In This Study
The neutral electrolyzed water was made immediately prior to the experiment.
After the generation of the electrolyzed-oxidizing water and prior to the commencement
of the washing and sanitization steps, the pH, free available chlorine (FAC) and
oxidation-reduction potential (Eh) of the EO water were tested. In this study, the pH
range was 6.5-7.5, the FAC was 100 ± 5 ppm and the Eh was 650-800 mV. The pH was
determined using a Hanna Instruments Microprocessor pH meter Model 210 (Hanna
Instruments Inc., Woonsocket, RI). The FAC concentration was measured with a HI
95771 Chlorine Ultra High Range Meter (Hanna Instruments, Ann Arbor, MI). Eh
measurements were obtained using a Mettler DL 70ES Titrator (Mettler Toledo,
Columbus, OH).
65
(2) PRO-SAN®
PRO-SAN® was supplied by Microcide, Inc. (Troy, MI) as a powdered
concentrate that required reconstitution before use. The recommended concentration for
sanitizing tableware and processing equipment was 10,000 ppm. The active sanitizing
agents in PRO-SAN® are citric acid (66%) and sodium dodecylbenzene sulfonate (3.6%).
Inert ingredients composed the remaining 30.4% of the powered concentrate.
(3) Quaternary Ammonium Compound (QAC)
The QAC used in this study had a brand name called Ster-Bac. It was produced
by Klenzade, a division of Ecolab, Inc. (St. Paul, MN). The main QAC found in Ster-Bac
was n-Alkyl [50% C14, 40% C12, 10% C16] dimethyl benzyl ammonium chloride and it
was used at a 200 ppm concentration. As mandated by the FDA Food Code (2005), the
use of QAC sanitizers must be mixed with water of not more than 500 ppm CaCO3. Thus,
before the use of Ster-Bac QAC, the water hardness was determined using a Water
Quality Test Strip kit (Hach Co., Loveland, CO) to ensure that this study conformed to
this mandate.
The water hardness for the mechanical and manual operations was
determined to be less than 120 ppm.
(4) Sodium Hypochlorite
The sodium hypochlorite solution was brand named “Chlor-Clean 12.5” and was
obtained from Madison Chemical Co., Inc. (Madison, IN). The concentration of the
sodium hypochlorite solution used in this study was 100 ppm.
66
2.3.5. Manual Warewashing Of The Tableware Items
The manual dishwasher was manufactured by Hobart Inc. (Troy, OH) and
consists of three compartments: (1) washing, (2) rinsing, and (3) sanitizing, as displayed
in Figure 2.4. The first compartment, the washing compartment, held approximately 60
gallons, while the rinse and sanitizer sinks had 40 gallon capacities. The tableware items
were washed with 100 ppm of the Mag Fusion detergent at 43°C for 20 s, rinsed with tapwater at 24°C for 5 s, and finally sanitized with different types of sanitizers at 24°C for 5
s. Prior to each experimental run, the manual dishwasher was thoroughly cleaned with
hot water and refilled with fresh water and detergent/sanitizer.
Washing
Sink
Sanitizing
Sink
Rinsing
Sink
Figure 2.4. Three-Compartment Turbowash Manual Warewasher Used In This Study
(Turbowash, Hobart, Inc., Tory, OH)
67
During the washing step, each contaminated tableware was washed manually
while wearing rubber gloves. A Scotch-Brite multi-purpose scrub sponge (3M, St. Paul,
MN) attached to a spring-loaded device was used to remove the cream cheese from plates
and plastic serving trays (Figure 2.5). The purpose of the spring-loaded was to provide
the same amount of force on each plate or tray being washed during the procedure. A
cylindrical device covered with a soft sponge was used to manually wash the drinking
glasses (Figure 2.5). The plastic trays were washed with four forward and four backward
strokes of the sponge. Plates and glasses were washed with directions of four clockwise
and four counter-clockwise strokes. These are methods adapted from Lee et. al (2007).
After the cleaning protocol, all tableware items were placed on a sterile rack and air-dried
for 1 hour at 24 ± 2°C before microbial testing.
Prior to the data collection, trial runs were performed in order to determine the
range of washing cycles that each sanitizer could handle and still produce the 5 log
bacterial reduction that the FDA Food Code mandates. Once this was determined, the
actual experimental procedures and data collection were taken. Each data point was an
average of 3 replicates.
68
Figure 2.5. Cylindrical Sponge (left) And Sponge Attached To The Spring-Loaded Test
Fixture (right) Used To Manually Clean Tableware Items.
2.3.4. Mechanical Warewashing Of The Tableware Items
The mechanical dishwasher used in this study was an AM Select Dishwasher
manufactured by Hobart Corp. (Troy, OH) (Figure 2.6).
This dishwasher had two
washing cycles: (1) washing and (2) sanitizing. The machine is a one-tank unit that
reuses the wash water in successive cycles and has a separate tank that holds the
sanitizing solution. The tableware items were washed with 1,000 ppm of the Guardian
Score (Ecolab, Inc., St. Paul, MN) detergent at 49°C (as per the manufacturer’s
recommendation) and sanitized with each of the 4 types of sanitizers at 24°C. Prior to the
use of the mechanical dishwasher, it was cleaned with hot water and refilled with fresh
detergent/sanitizer solutions.
69
The wash water was sprayed onto the dishes for 40 sec at a pressure of 20 psi.
Spraying of the detergent solution occurred from both top and bottom slotted pipes within
the washer. Subsequently, the tableware was sprayed with fresh sanitizing solution for
10 sec. After the sanitization cycle, all tableware items were air-dried for 1 hour at 24 ±
2°C on a sterile surface prior to sampling. Similar to what was done for the manual
washing procedure, trial runs were carried out in order to determine the range of
warewashing cycles for tableware/sanitizer that will produce the 5-log bacterial reduction
mandate by the FDA Food Code.
Figure 2.6. AM Select Mechanical Washer Used In This Study (Hobart, Corp.)
70
2.3.6 Microbial Enumeration Of The Contaminated Tableware Surfaces
After air-drying the tableware for approximately 1 hour, sterile calcium-alginate
cotton-tipped swabs (Fisher Scientific, Pittsburgh, PA) were used to transfer any
microorganisms that were left on the surface of the plates and drinking glasses. These
swabs were moistened with sterile maximum recovery diluent (MRD) (Oxoid Ltd.,
Basingstoke, Hampshire, England) before swabbing. After swabbing the tableware, the
swabs were placed in a tube containing 2 mL of sterile MRD. These tubes were vortexed
to remove any bacterial cells that were attached to the tip of the swab. The cotton swabs
were then aseptically removed and the contents of the test tubes serially diluted and
plated onto TSAYE. They were then incubated at 37°C for 36 h. The bacteria cells were
counted using a Darkfield colony counter (American Optical, Buffalo, NY).
The
detection limit for estimating the bacteria survival was 20 CFU/tableware for plates and
trays, and 2 CFU/tableware for the drinking glasses.
A sterile sponge swab was used to sample the plastic serving trays. These sterile
sponges were provided in a stomacher bag by Fisher Scientific (Pittsburgh, PA). Prior to
sampling, each sponge was moistened with 5 mL of MRD. The sponges were then
pressed, using finger pressure, along the entire surface of plastic trays. Each sponge was
placed back into the stomacher bag and a 45 mL aliquot of MRD was added and the bag
massaged for 2 min using a Biomaster 80 Stomacher (Seward Laboratory, London, UK).
Serial dilutions were then performed and plated onto TSAYE and incubated at 37°C for
36 h.
71
2.3.7. Statistical Analysis
All trials were repeated three times in this study.
Microbial counts were
expressed as log CFU per milliliter (inoculum) and log CFU per tableware (surface).
During each trial, a randomized number generator was used to randomly select 3
tableware items for bacterial enumeration. The reported values were the means of the
number of washing cycles that each sanitizer can handle and still produce a 5-log
bacterial reduction.
Analysis of variance (ANOVA) was used to determine the
significance between the means when considering: (1) the type of sanitizer used, (2) the
type of tableware item used, (3) the type of protocol (manual vs. mechanical) used and (4)
the type of bacterial species used.
If ANOVA found a significant effect, multiple
comparison testing was performed (Bonferroni and Tukey HSD) to determine which
treatments were significantly different from each other and to rank them. Statistical
testing was performed by using SPSS Version 16.0 (SPSS, Inc., Chicago, IL). A p-value
of 0.05 was set for the level of significance.
2.4. Results and Discussion
2.4.1. Effect Of Air-Drying On The Reduction Of The Microbial Population
In order to simulate normal foodservice operations, the tableware items were
allowed to air-dry for 1 hour after the food contaminated were applied to them. This
drying period also allowed enough contact time of the food soil to adhere to the tableware
items. Table 2.1 shows the effect of this drying time on the reduction of the microbial
population. The mean reduction of both L. innocua and E. coli K12 that were inoculated
into the cream cheese was ~ 0.30 log CFU/tableware. For the contaminated milk samples,
72
there was a decrease of ~ < 0.10 log CFU/tableware. The statistical analysis showed that
there were no significant differences (P > 0.05) between the bacterial populations before
and after the contaminated food products were dried on the various tableware items.
These results were similar to results obtained by Lee et al. (2007) and Handojo et al.
(2009) when they studied tableware washing protocols.
Table 2.1. Bacteria Survival (log10 CFU/tableware) On Various Contaminated Tableware
Before And After 1 hour Air-Drying At 24°C, Prior To The Washing And Sanitization
Protocol.
Cream Cheese
Drying
Escherichia coli K12
Listeria innocua
Milk
Plate
Tray
Glass
Before
9.30±0.40
9.54±0.60
9.01±0.30
After
9.00±0.40
9.19±0.70
8.98±0.20
Before
9.30±0.40
9.53±0.80
9.02±0.30
After
9.01±0.50
9.20±0.70
8.93±0.80
2.4.2. Efficacy Of The Various Sanitizing Agents Used In This Study
The washing and sanitization protocol used in this study conformed to the
mandates that are stated in the FDA Food Code (2005). For mechanical warewashing,
49°C (120°F) and a spray-pressure of 138 KPa were used for all experiments. During the
manual protocol, the washing variability was minimized by having the same individual
clean the tableware items for all experimental runs.
Figure 2.7 shows the maximum number of warewashing cycles that produced a 5log bacterial reduction of L. innocua with a single batch of cleaning compounds
(detergent, rinse water and sanitizer). Figure 2.8 also displays the same results, but with
73
E. coli K12 as the contaminating bacterial species. The results show that there were
significant difference (P < 0.05) (Tukey’s HSD and Bonferroni) between NEW and PROSAN® and the other traditional sanitizers (sodium hypochlorite and QAC). The statistics
also showed that there was no statistical difference (P > 0.05) between the efficiency of
# o f W ash in g Cycles
sodium hypochlorite and QAC.
24
22
20
18
16
14
12
10
8
6
4
2
0
Trays Plates Glass Trays Plates Glass Trays Plates Glass Trays Plates Glass
NEW
PRO-SAN
CHLORINE
MANUAL WASHING
QAC
MECHANICAL WASHING
Figure 2.7. The Maximum Number Of Warewashing Cycles That Can Produce A 5-Log
Bacterial Reduction Of Listeria innocua With A Single Batch Of Detergent, Rinse Water
And Sanitizer.
74
# o f W ash in g Cycles
20
18
16
14
12
10
8
6
4
2
0
Trays Plates Glass Trays Plates Glass Trays Plates Glass Trays Plates Glass
NEW
PRO-SAN
CHLORINE
MANUAL WASHING
QAC
MECHANICAL WASHING
Figure 2.8. The Maximum Number Of Warewashing Cycles That Can Produce A 5-log
Bacterial Reduction Of Escherichia coli K12 With A Single Batch Of Detergent, Rinse
Water And Sanitizer.
As more tableware items were washed in the same batch of cleaning chemicals,
the sanitizing efficacy ultimately began diminshing. This was due to organic matter that
built up in the cleaning solutions. This organic matter usually reacts with the active
ingredients responsible for biocidal action of the cleaning chemicals (Ruiz-Cruz et al.,
2007). Consequently, the concentration of these active ingredients would diminish as
more tableware items are washed and sanitized. Based on results from this study, the
efficacies of the NEW and PRO-SAN® sanitizers were greater than the traditional
sanitizers. This information is important for foodservice establishments, not only for
cost-saving reasons, but the newer sanitizers are less harmful to employees and have a
lower negative impact for the environment.
75
The inclusion of the surfactant (sodium dodecylbenzene sulfonate) and citric acid
in the PRO-SAN® formulation gave it an advantage over the other sanitizers. Surfactants
act by increasing the “wetting” properties of a solution and this enhances its capability to
cause the release of water-insoluble organic compounds that are bound to a food-contact
surface (Neupane and Park, 1999). Once the organic matter is released, the citric acid
acts to reduce the microbial population. Citric acid is also a sequestering agent and it
binds to metal ions that are attached to the contact surface and makes the metal ionic
compound water soluble (Lee et al., 2007). As a result, the microbial cells that were once
shielded by the metal ion complexes are now exposed to the acidic solution. PRO-SAN®
also has the advantage of having ingredients that are natural, biodegradable and do not
produce carcinogenic by-products or free radicals. However, one disadvantage is that
PRO-SAN® produces a large amount of foam and leaves a film-like residue on the
tableware surface after sanitization protocols (Handojo et al., 2009). Therefore, there
may be a need for an additional rinsing step with this sanitizer.
The NEW sanitizer was also more efficient than the traditional sanitizers mainly
due to its high Oxidation-Reduction Potential (Eh) and the free-available chlorine (FAC)
it contained. The Eh value for the NEW solutions used in this study was between ~ +700850 mV and the FAC ranged from 95-105 ppm. This high Eh value indicated that the
sanitizing solution sequestered electrons from the cellular membranes of microorganisms.
This allowed the FAC to enter the cytoplasm and inactivate the cellular metabolic
functions (Kim et. al, 2000). Since the solution is at neutral pH, it was non-corrosive to
processing and warewashing equipment. Other advantages of NEW include the fact that
it was less hazardous to workers and the environment. This is due to the fact that the
76
sanitizer could be produced on-site, immediately before use, thus minimizing the
transport and storage of concentrated chemicals. In addition, after the initial investment
of the EO-Water generator, the only operating costs are the tap water, sodium chloride
and routine maintenance (Walker et al., 2005).
Improper washing and sanitizing protocols or trying to extend the use of a single
batch of cleaning and sanitizing chemicals can increase the risk of cross-contamination to
ready-to-eat foods. Bloomfield and Scott (1997) reported that ingestion or contact with
relatively small numbers of pathogenic microorganisms could be adequate to cause
infection, especially with E. coli O157:H7. Consequently, several ways to decrease this
health risk, including: (1) complete removal of food soil from food-contact surfaces, (2)
air-drying after washing and a (3) personal hygiene program (Wernesson et al., 2004;
Hirai, 1991; Bloomfield and Scott, 1997; Kusumaningrum et. al, 2002; Cogan et al.,
1999).
2.4.3. Comparative Efficiencies Of Manual With Mechanical Warewashing
Operations
As displayed in Figs 2.7 and 2.8., mechanical warewashing was more efficient
than manual washing operations. The statistical analysis (Tukey HSD and Bonferroni)
confirmed this statement, showing that mechanical was statistically (P < 0.05) more
effective than manual warewashing. This result is confirmed by previous studies done by
Ebner et al, (2000), who found that mechanical warewashers produces a consistently high
standard of cleaning and disinfection.
Manual warewashing, on the other hand, is
dependent on the experience and knowledge of the employee, combined with the use of
77
proper cleaning and sanitization protocols. In fact, a study by Wernersson et al. (2003)
concluded that mechanical washers control the microbiological level better than the most
basic level of manual dishwashing. Usually, manual cleaning of tableware items in
foodservice establishments are performed by employees with low compensation and
minimal opportunity for career advancement. These employees may feel less inclined to
perform their duties at the minimum required standard and this may lead to improperly
washed tableware items.
As discussed earlier, this study strictly followed the proper washing and sanitizing
protocol as outlined in the Food Code. The temperature was constantly monitored to
conform to this standard, but in real-life situations at foodservice establishments,
tableware cleaning at the minimum required temperature may not always occur. Mattick
et al. (2003) confirmed this in studies they performed. In those studies they focused on
the microbiological quality of the cleaning water used by volunteers during a tableware
washing exercise.
The results found that cleaning water >40°C (>104°F) had
significantly lower aerobic plate counts than cleaning water <40°C. According to Pfund
(2004), temperatures ≥40°C may be uncomfortable for dishwashing employees and thus
this minimum temperature mandate is not always met. Mechanical washing, conversely,
usually uses higher washing temperature (55-65°C) (Wernersson et al., 2006). Other
factors that vary the efficiency of manual washing are the number and sizes of the
tableware items to be washed, the initial microbial load, the type of food soil
contaminating the tableware items and the time designated to clean them (Montville et al.,
2002; Mattick et al., 2003; Lee et al., 2007).
78
2.4.4. Effect Of Material Type On The Efficiency Of Washing And Sanitizing
Protocols
The results showed significant differences (P < 0.05) in the efficiencies of
washing and sanitizing protocols when the influence of material type was considered.
Based on Figs. 2.7 and 2.8., the material types harboring the highest microbial loads were
plastic trays > ceramic plates > glasses. This result may have occurred because of the
surface wear and subsequent pitting of the material. This surface wear will affect the
subsequent hygienic and cleanability status of the tableware, as it affects its topography
and chemistry, thus a measure of the type and degree of its roughness is essential (Verran
et al., 2000). Figures 2.7 and 2.8 show that plastic trays were the least efficient to clean
due in part to its food contact surface that had the roughest design when compared with
the ceramic plates and drinking glasses. In addition to this, plastics are usually soft and
relatively easy to scratch. It is this reason why plastic materials are not preferred for use
as food contact surfaces during processing, although for cost-saving reasons they may
find many uses, particularly in the home as containers, cutting boards, waste bins and
other applications (Verran et al., 2000). After a few uses, plastic material may develop
crevices large enough to harbor bacteria (Bower et al., 1996). These crevices could act to
shield the biocidal action of sanitizing solutions, which require direct contact with
bacterial cellular membranes. These may have also occurred in this study because of the
differences in surface area of the tableware items. Trays had the largest surface area,
followed by plates and lastly drinking glasses. Table 2.1 shows that the plastic trays had
the highest initial microbial load prior to commencing the washing protocol and this may
be the reason why this type of tableware and material was the least efficient.
79
Another factor that may have affected the results is the difference in the two types
of food samples used in this study. Most components of cream cheese are non-water
soluble, while a majority of the components in whole milk are water-soluble. After the 1
h drying time to allow the inoculated food samples to adhere to the tableware surface, the
cream cheese appeared “baked-on” and was difficult to manually remove. Solid fat
deposits are water-immiscible, thus making them more difficult to remove when adhered
to a surface. Milk, in contrast, contains less fat and is soluble in water, making it easier
to remove from food-contact surfaces (Schmidt, 2003).
2.4.5. Survivability of Listeria innocua And Escherichia coli K12 After Washing And
Sanitizing Protocols
The health risks associated with E. coli O157:H7 and L. monocytogenes are well
known. E. coli O157:H7 causes approximately 73,000 illnesses and 61 deaths per year
(Mead et al., 1999). L. monocytogenes is a pathogen that is found ubiquitously in the
environment.
These species are even found in food-processing settings even after
performing cleaning and sanitization of food contact surfaces (Taormina and Beuchat,
2002). L. innocua and E. coli K12 were chosen as surrogates to the pathogenic strains
because the washing studies were performed in a pilot plant which food is prepared. The
use of surrogates has been proven to be a viable alternative to pathogen testing (MoceLivina et al., 2003).
Additionally, we chose these bacterial species to compare the
survivability of Gram-positive species (Listeria) with that of Gram-negative ones
(Escherichia). Based on the results obtained (Figs 2.7 and 2.8), there was no significant
(P > 0.05) difference in the survivability of the two bacterial species tested. Nonetheless,
80
it is very important to correctly perform washing protocols since both E. coli O157:H7
and L. innocua only requires very few cells per gram or mL to be infectious and are able
to survive certain washing processes and form biofilms (Bower et al., 1996).
2.5. Conclusions
This study demonstrated that NEW and PRO-SAN® are more efficient than
traditionally used sanitizers for bacterial reduction on tableware items.
Glassware
produced the highest maximum number of washing cycles, while plastic trays produced
the lowest maximum. Also, mechanical washing operations was more efficient than
manual washing. These newer sanitizers can be used by the foodservice industry to
reduce costs, while also being less hazardous to employees and environmentally-friendly.
81
CHAPTER 3
A COMPARISON OF VARIOUS CHEMICAL SANITZERS IN THE REMOVAL
OF ORGANIC MATTER ON GLASS SURFACES BY ATOMIC FORCE
MICROSCOPY
3.1. Abstract
Improper washing and sanitizing of tableware items is a significant public health
risk. Residual food soil that is left on food-contact surfaces can be used by pathogenic
microorganisms for growth, replication and may even allow them to form biofilms. This
study investigated the effect of various sanitizers (sodium hypochlorite, quaternary
ammonium, neutral electrolyzed water and PRO-SAN®) on removing various milk-based
products (whole, 2% reduced fat, chocolate low fat and skim milk) from underlining
glass surfaces. Atomic force microscopy was used to determine the thicknesses of the
milk-films left after attempts to clean the glass surfaces. Results showed that PRO-SAN®
significantly reduced the amount of residual food soil when compared with the other
sanitizers. This was due sodium dodecylbenzene sulfonate (a surfactant) being present in
the PRO-SAN® formulation. The data also showed that whole and chocolate milk would
be more difficult to clean when compared with 2% and skim milk.
82
3.2. Introduction
Residual food left on eating utensils and processing equipment could be a safety
hazard if not properly cleaned but used to prepare or serve ready-to-eat foods. To
minimize this risk, the Food Code (2005) and NSF International Standards mandate that a
5 log bacterial reduction must be achieved from washing protocols on eating utensils, and
that all “old food” must be visually removed. If this is not adequately done, pathogenic
microorganisms of public health concern can readily use the organic matter in the food as
nutrients for growth and replication and subsequently cross-contaminate ready-to-eat
foods. Additionally, if certain pathogens remain on a given surface for a relatively long
period of time, they can continue to replicate, attach to the surface and eventually form
biofilms (Uhlich et. al, 2006). This leads to even greater health related issues (Kumar
and Anand, 1998; Venter, et al., 2006). It is thus essential to effectively remove all food
films when performing washing protocols by using the correct type of detergent and/or
sanitizer. The correct selection of these agents would assist in removing all residual food
soil at the end of the cleaning protocol.
This chapter discusses the effectiveness of four chemical sanitizers (sodium
hypochlorite, QAC, PRO-SAN® and EO water) by focusing on their abilities to remove
various organic matter residues in the form of food soils. The choice of test utensils and
contaminating food types were carefully chosen so that a worst-case scenario was created.
This will ensure that easier to clean tableware items can be adequately cleaned and
sanitized. Based on previous studies, milk-based products that were left on drinking
glassware were found to be the most problematic to clean and harbored the highest
bacterial load when compared with other types of foods (Lee et al., 2007; Handojo et al.,
83
2009). Additionally, Schmidt (2003) stated that food proteins are the most difficult soil
to remove during washing protocols, and that milk caseins are used for its adhesive
properties in glue and paint products. Consequently, various milk products were chosen
for this experiment.
The milk products used were: (1) whole milk, (2) 2% reduced fat milk, (3)
chocolate milk and (4) skim milk. Each of these milk products are composed of various
dissolved solids and suspensions (USDA, 2007). These include compounds such as
proteins, minerals, phospholipids, sugars and salts (Michalski and Briard, 2003). Milk
products can be differentiated by its water and fat content. Skim milk contains the
highest percentage of water (90.8%), while having the lowest fat content (0.2%). Twopercent reduced fat milk contains 89.3% water and 2.1% fat, while chocolate milk
contains 84.5% water and 1.0% fat. Lastly, whole milk has 88.3% water and 3.3% fat.
In studies done by Handojo et al. (2009), they found that whole milk was the
hardest to clean, followed by chocolate milk, 2% milk and lastly skim milk. They also
showed that atomic force microscopy (AFM) is a reliable and alternative method for
estimating the adhesion forces between milk-based samples and underlining surfaces.
This is this reason why AFM was used in this study.
The objectives of this study were: (1) to compare the ability of selected sanitizers
to remove different milk-based products from glass surfaces after a worse-case scenario
washing protocol and; (2) to confirm whether AFM has the ability to perform this
analysis.
84
3.3. Materials and Methods
3.3.1. Food Sample Preparation
Whole, 2% reduced fat, chocolate low fat and skim milk-based products were
purchased from a local grocery store (Columbus, OH) one day prior to each experimental
run. They were stored in an incubator at 4 ± 1°C. These milk samples were used to soil
glass surfaces for the purpose of measuring the ability of AFM to test the effectiveness of
the washing protocols.
The test was initiated by placing a 200 µl drop of each type of milk product on
separate pre-cleaned glass microscope slides (the microscope slides simulated the
surfaces of drinking glasses). These were dried for 1 h at 24 ± 3°C to allow for the
organic matter to sufficiently adhere to the glass surface, as well as simulating normal
food-service operations. One replication of testing consisted of 8 microscope glass slides
(2 slides with a drop of either whole, 2% reduced fat, chocolate, or skim milk-based
products) (Figure 3.1.), according to a method developed by Handojo et al. (2009).
85
Figure 3.1. A Photograph Of The Slides Used In This Study
3.3.2. Modified Washing Protocol
After preparing the sample microscope slides, each replicate was then washed
using a mechanical warewasher provided by Hobart Corp. (Troy, OH). The detergent
used for this procedure was Guardian Score (Ecolab, Inc., St. Paul, MN).
The
manufacturer recommends the usage of the detergent at a 1,000 ppm concentration and
the FDA Food Code (2005) recommends a washing temperature of at least 49°C.
86
However, in this study, the washing procedure was modified to create a “worst-case”
scenario. For this experiment, the detergent concentration and washing temperature was
500 ppm and 35°C, respectively. This modified protocol left a residual milk film on the
glass surface and this allowed for the use of AFM to determine the thickness of this milk
film.
After the washing step, each set of slides was sprayed with one of the chemical
sanitizers for 10 seconds in the mechanical warewasher. The test was repeated for each
of the different sanitizers. The four sanitizers used in this experiment were (1) sodium
hypochlorite (Chlor-clean 12.5, Madison, IN) at 100 ppm, (2) Quaternary Ammonium
Compound (Ster-Bac, St. Paul, MN) at 200 ppm, (3) an organic acid (PRO-SAN®, Troy,
MI) and (4) neutral electrolyzed-oxidizing water (Ecaflo 110, Trustwater Inc., Ireland) at
100 ppm free available chlorine. The slides were subsequently dried for 1 hour at room
temperature before proceeding to the AFM analysis. Each replication was repeated three
times for each of the four sanitizers used in this study.
3.3.3. Atomic Force Microscopy Measurements
The AFM analysis was carried out using a Nanoscope III-Dimension 3100
microscope (Digital Instruments, Santa Barbara, CA) at room temperature (24 ± 2°C).
The AFM machine used in this analysis is displayed in Figure 3.2. The AFM nanoprobe
cantilever used was a 0.5-2.0 Ω cm phosphorous doped silicant, with a nominal spring
constant of 3 N/m and obtained from Veeco Instrument (Santa Babara, CA, CA). The
length of the cantilever was 215-235 µm. The AFM tips were provided by Veeco Probes
87
(Camarillo, CA). They were coated with silicon and had a nominal spring constant of 3
N/m, a nominal frequency of 75 KHz and a tip height of 15-20 µm (Figure 3.3).
Figure 3.2. AFM Machine Used In This Experiment (Digital Instruments, Santa Barbara,
CA).
88
CANTILEVER
AFM TIP
SAMPLE
SURFACE
Figure 3.3. Scanning Electron Microscope Image Of An AFM Tip Used To Probe The
Structure Of The Sample Surface. 10,000x Magnification (Morris et. al, 1999).
During the AFM test, the contact mode, also known as the scratching mode, was
used to remove a 5 x 5 µm area of each milk-based sample from the underlying glass
surface. This procedure was performed using the following AFM equipment settings:
•
Sum score between 4-6
•
Horizontal deflection set at 0
•
Vertical deflection set as -2
•
Scan rate – at 2.00 Hz
The force applied to each sample was approximately 1-5 V. The force required to
completely remove the milk-based film from the glass surface was recorded. The applied
force was adjusted when necessary depending on the type of milk product that was being
tested.
89
Once the 5 x 5 µm film was adequately and evenly scratched, the resulting surface
was then imaged and further analyzed using the AFM tapping mode. This tapping mode
provided a 3-D topographical image that was produced using the computer software
connected to the AFM instrument.
The thickness of the organic matter was then
determined from the image produced by the computer software.
Each slide was
analyzed at three different areas and the data collected were averaged.
3.3.4. Statistical Analysis
All reported values are the means of three replicates and the test was performed
twice. Statistical analysis of variance (ANOVA) was first used to determine overall
significant differences, followed by post-hoc (Bonferroni and Tukey HSD) tests using
SPSS Version 16.0 (SPSS, Inc., Chicago, IL). A p-value of 0.05 was set for the level of
significance.
Tukey’s HSD and Bonferroni tests were used to analyze the film
thicknesses of various milk samples after cleaning protocols with various sanitizing
agents.
3.4. Results and Discussion
3.4.1. Force Required To Remove A Selected Milk-Based Film Area During The
AFM Scratching Test.
Table 3.1 presents the average force required to remove the residual milk-based
films from glass surfaces after the washing protocol with the selected sanitizers. This
data represents the force of the AFM scratching test. This table shows that whole and
chocolate low-fat milk samples required more downward force on the cantilever to
90
completely remove the residual films from glass surfaces. The 2% reduced and skim
milk samples required less force to achieve the same result.
Table 3.1. The Force (V) Required To Completely Remove Various Milk-Based Samples
From The Glass Surfaces After Washing Protocols With Various Sanitizers
Whole
Chocolate
2% Reduced
Skim
Sodium
Hypochlorite
4.61 ± 0.15
4.02 ± 0.33
3.29 ± 0.13
2.51 ± 0.13
Quaternary
Ammonium
4.66 ± 0.14
3.86 ± 0.32
2.96 ± 0.13
2.46 ± 0.21
PRO-SAN®
EO-Water
3.32 ± 0.21
2.93 ± 0.16
2.40 ± 0.13
1.31 ± 0.15
4.78 ± 0.21
4.29 ± 0.10
2.92 ± 0.12
2.47 ± 0.12
This result occurred because of the differences in moisture content of the four
types of milk-based products used in this study.
Pure water will adhere less to
underlining surfaces when compared with non-homogenous liquids with lower moisture
contents, including milk-based products. Milk products are emulsified colloids of liquid
butterfat globules dispersed within a water based fluid. They are also composed of
several dissolved and suspended solids, including proteins, fats, sugars and salts (Jensen,
1995; Michalski et al., 1999; Dickinson, 2001). The inclusion of these solids in milkbased products lowers the moisture content. As previously discussed, skim milk has the
highest moisture content (90.8%), followed by 2% reduced fat, whole and lastly
chocolate low-fat milk. Water is known to act as a plasticizer for organic matter and will
act to separate the bonding between the milk films and the glass surface (Adhikari et al.,
2001). Therefore, less force is required to remove skim milk-based films when compare
with those having a lower moisture content.
91
The results displayed in Table 3.1
corroborated this inverse relationship of moisture content and strength of adherence to
surfaces, except for the chocolate milk samples.
Chocolate low-fat milk has the lowest moisture content due to the extra
ingredients not found in the other milk-based samples. These include high fructose corn
syrup, cocoa powder, corn starch and carrageenan that are distributed within a continuous
cocoa butter phase (Hodge and Rousseau, 2002). It would be hypothesized that due to
the complexity of the chocolate milk solution that it would be the hardest to remove and
have the thickest residual film after the cleaning protocol. This was not the cases, as
whole milk samples required the highest force to completely remove the 5 x 5 µm milkbased film area.
The greater force required to remove the whole milk may be due to its fat content
having a higher adherence to the glass surfaces.
A study by Rennie et al. (1999)
compared the cohesion of whole and skim milk powders (at the same moisture content
and temperature) to underlining contact surfaces. Results of their study showed that the
cohesion of whole milk powder was almost twice that of the skim milk powder.
Furthermore, milk fat inhibited the diffusion of moisture from the interior of the drop to
the surface for evaporation, and this allowed more time for attraction at the liquid-liquid,
liquid-solid and liquid-solid interfaces (Stevenson et al., 1998; Michalski et al., 1997).
Milk proteins also play a role in adhesion to underlining surfaces, but the milk types used
in this study contained similar protein contents (17%) and would not have influenced the
results.
92
3.4.2. Milk-Based Film Thickness Data Of Traditional Sanitizers (Sodium
Hypochlorite And QAC)
Figure 3.4 displays a 3-D tapping mode image (12.5 x 12.5 µm area) of a
scratched portion of a whole milk sample after being washed and sprayed with sodium
hypochlorite. Figure 3.5 shows the cross-sectional analysis of the same whole milk
sample.
This cross-sectional analysis provided information regarding the average
thickness of the milk film. Similar 3-D images and cross-sectional analyses (not shown
in this paper) were obtained for each of the other milk-based samples used in this study.
Figure 3.4. A Tapping Mode 3-D Topographical AFM Image Of The Surface Of A
Whole Milk Sample After The Washing Protocol With Sodium Hypochlorite.
93
Figure 3.5. Cross-Sectional Analysis Of The Scratched Area Of The Whole Milk Sample
On The Glass Surface.
Figures 3.6 and 3.7. report the average residual film thicknesses for whole, 2%
reduced fat, chocolate low-fat and skim milk on glass surfaces after the washing protocol
with sodium hypochlorite and QAC, respectively. These figures show that the order of
thickness of the various milk samples left on the glass surfaces from highest to lowest
were whole, chocolate low-fat, 2% reduced fat and skim milk, respectively. Statistical
analyses (Tukey’s HSD and Bonerroni) confirmed these results as being significantly
different in thickness (P < 0.05) between all milk samples after the washing protocol
QAC.
For the washing protocol using sodium hypochlorite, there were significant
differences on the thicknesses between all samples except between the 2% reduced fat
and chocolate low-fat milk samples. There were no significant differences (P >0.05)
between the thicknesses of 2% reduced fat and the chocolate low-fat milk samples.
These results support the data displayed in Table 3.1, where the whole milk samples
94
required the greatest downward force to remove the residual films on the surface of the
glass slide. The results also show that there was not a significant difference (P >0.05) in
the film thicknesses when the hypochlorite compared with the QAC sanitizer were used
to clean the slides.
160
Film Thickness (nm)
140
120
100
80
60
40
20
0
Whole Milk
Chocolate Milk 2 Percent Milk
Skim Milk
Figure 3.6. Mean Thickness (nm) Measurements By AFM Analysis Of Various Milk
Products After Washing Protocols Using Sodium Hypochlorite.
95
180
Film Thickness (nm)
160
140
120
100
80
60
40
20
0
Whole Milk
Chocolate Milk 2 Percent Milk
Skim Milk
Figure 3.7. Mean Thickness (nm) By AFM Analysis Of Various Milk Products After
Washing Protocols Using QAC.
3.4.3. Milk-Based Film Thickness Data Of Novel Sanitizers (EO-WATER and PROSAN®)
Figures 3.8 and 3.9. report the average residual film thicknesses for whole, 2%
reduced fat, chocolate low-fat and skim milk on glass surfaces after the washing protocol
with EO-Water and PRO-SAN®, respectively. These results are similar to those obtained
for the traditional sanitizers. The results show that there were significant (P < 0.05)
differences in film thicknesses of the four milk-based products tested. Figure 3.9 also
shows that more milk film was removed after the modified washing protocol and the use
of PRO-SAN®. Tukey’s HSD and Bonferroni statistical analyses confirmed that PROSAN® had a greater effect in removing organic matter from food contact surfaces than the
other three sanitizers tested in this study. This is due to the sodium dodecylbenzene
sulfonate being present in the PRO-SAN® formulation. This compound is a surfactant
96
which lowers the surface tension of liquids. By lowering the surface tension a higher
“wetting” of the soil particles occurs, and this increases its detachment from the contact
surface. This result signifies that PRO-SAN® has the extra ability of removing organic
soil from a food-contact surface that may be left after inadequate washing that the other
sanitizers do not possess.
180
160
Film Thickness (nm)
140
120
100
80
60
40
20
0
Whole Milk
Chocolate Milk
2 Percent Milk
Skim Milk
Figure 3.8. Mean Thickness (nm) By AFM Analysis Of Various Milk Products After
Washing Protocols Using EO-Water.
97
140
Film Thickness (nm)
120
100
80
60
40
20
0
Whole Milk
Chocolate Milk 2 Percent Milk
Skim Milk
Figure 3.9. Mean Thickness (nm) By AFM Analysis Of Various Milk Products After
Washing Protocols Using PRO-SAN®.
Figures 3.9, 3.10, 3.11 and 3.12 confirms the conclusion that using PRO-SAN®
better removes food soil that has adhered to underlining surfaces. All figures illustrates
that PRO-SAN® had the lowest film thickness with all milk types.
This data was
corroborated with statistical testing showing that using PRO-SAN® had a significant (P <
0.05) effect in removing milk film when compared to the other sanitizers.
98
180
Film Thickness (nm)
160
140
120
100
80
60
40
20
0
Chlorine
QAC
NEW
PRO-SAN
Figure 3.10. Mean Thickness (nm) By AFM Analysis Of Whole Milk After Washing
Protocols Using Various Sanitizers.
Film Thickness (nm)
120
100
80
60
40
20
0
Chlorine
QAC
NEW
PRO-SAN
Figure 3.11. Mean Thickness (nm) By AFM Analysis Of Chocolate Milk After Washing
Protocols Using Various Sanitizers.
99
90
Film Thickness (nm)
80
70
60
50
40
30
20
10
0
Chlorine
QAC
NEW
PRO-SAN
Figure 3.12. Mean Thickness (nm) By AFM Analysis Of 2 Percent Reduced Fat Milk
After Washing Protocols Using Various Sanitizers.
70
Film Thickness (nm)
60
50
40
30
20
10
0
Chlorine
QAC
NEW
PRO-SAN
Figure 3.13. Mean thickness (nm) by AFM analysis of skim milk after washing protocols
using various sanitizers.
100
3.4.4. Confirmation of AFM As A Reliable Analytical Procedure For Food Samples
AFM is a technique used to measure and produce a high resolution threedimensional image of extremely small particles, in the nano-scale, that other analytical
techniques cannot provide (Binnig et al., 1987; Herrmann, et al., 2004). In recent years,
the use of AFM expanded to analyzing the structure of various food items (Morris et al,
2001, Morris, 2004). A study by Handojo et al. (2009) measured the adhesion strengths
of various milk products on glass surfaces using traditional methods (contact angle and
surface tension) and an alternative method, AFM. Their study concluded that AFM was
an alternative option when measuring and differentiating the adhesion strengths of
various soft food samples. This study also confirmed the capability of AFM analyzing
adhesion strengths of various milk-based products on glass surfaces.
The reason why AFM can differentiate extremely small particles is due to the
tapping mode function. This tapping mode is a useful AFM method for imaging a
deposit of milk based solids on to a glass surface. Since the milk based solids are not
covalently bonded to the glass, the AFM method must image them without disturbing the
arrangements of the milk molecules. The tapping mode makes this possible since it
images the surface of the milk-based particles as it oscillates at an amplitude that
corresponds to the topography of the surface (Putman et al., 1994). As the tip oscillates
close to the surface, forces such as Van der Waals, capillary, electrostatic and magnetic
forces between the molecules on the tip and the sample leads to deflections of the
cantilever (Allen et al., 1997; Smith, 1999). Concurrently, the laser beam focuses on the
surface of the cantilever, and the deflections it relays to the photodiode goes to a detector
101
which ultimately creates a topical three-dimensional surface image. The AFM software
then calculates the thickness of the residual milk films on the glass surface. As a result,
the effectiveness of the sanitizers that were tested can be determined by comparing the
level of milk film thickness left on the glass surface.
3.5. Conclusions
This study confirmed that AFM can be used to determine the thickness of a thin
film of milk left on a drinking glass after a washing protocol that includes a chemical
sanitizing spray. The thickness of the film could be used to estimate the difficulty of
removing milk from drinking glasses. This study also corroborated the results from a
previous study that whole milk and chocolate milk had the strongest adherence to a glass
surface as compared to 2% reduced fat and skim milk.
Furthermore, this study
demonstrated that the sanitizer PRO-SAN® has the ability to remove organic matter that
has adhered to a glass surface, more so than the other sanitizers that were tested in this
study.
102
CHAPTER 4
CONCLUSION
This study demonstrated that NEW and PRO-SAN® could be used as effective
chemical sanitizers for reducing pathogens of public health concern on various tableware
items in both manual and mechanical warewashing operations. These novel sanitizers
also showed that they are more efficient than two traditionally used ones (sodium
hypochlorite and QAC).
Also illustrated was the fact that mechanical was more
economical than manual warewashing.
This study showed that the type of tableware
material (plastic vs. ceramic vs. glass) played a role in the washing efficiency. This
information could be used by the foodservice industry as a cost-savings measure. Also
the newer sanitizers have a lower negative impact on employees and the environment.
A supplemental study concluded that AFM can be used to measure the thickness
of food soil that had adhered to underlining glass contact surfaces. These measurements
can then be used to determine the adhesion strength of the food soil to the surface. This
additional study demonstrated that whole and chocolate milk had the strongest adhesion
to the glass surface, when compared with 2% reduced fat and skim milk. Furthermore,
PRO-SAN® demonstrated that it was the most effective in removing food soils from a
103
glass surface due to the inclusion of a surfactant (sodium dodecylbenzene sulfonate) in
the product formulation.
104
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(ETEC).
APPENDIX A: RAW DATA
120
EO
WATER
Plates
Trays
Glass
PRO-SAN
Plates
Trays
Glass
CHLORINE
Plates
Trays
Glass
QUAT
Plates
Trays
Glass
MANUAL
WASHING
AUTOMATIC
WASHING
10
9
9
9
9
9
15
15
17
10
11
10
8
8
9
16
15
16
8
9
9
7
7
7
14
16
15
10
10
12
7
8
8
15
15
16
12
12
12
9
9
10
19
19
21
11
12
12
9
11
9
21
21
19
10
11
10
9
10
9
17
17
17
11
11
11
7
7
8
17
19
17
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Table A.1. Raw Data For The Maximum Number Of Warewashing Cycles That Can
Produce A 5-Log Bacterial Reduction Of Listeria innocua With A Single Batch Of
Detergent, Rinse Water And Sanitizer.
121
EO WATER
Plates
Trays
Glass
PRO-SAN
Plates
Trays
Glass
CHLORINE
Plates
Trays
Glass
QUAT
Plates
Trays
Glass
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
Rep 1
Rep 2
Rep 3
MANUAL
WASHING
10
10
11
7
7
7
15
16
16
10
11
10
9
9
9
17
16
16
8
9
9
6
7
6
14
15
15
9
10
10
6
7
6
15
14
15
AUTOMATIC
WASHING
11
11
12
7
7
8
16
19
18
13
12
12
10
10
11
18
19
19
9
11
10
9
9
10
18
16
16
12
12
12
9
8
8
17
16
19
Table A.2. Raw Data For The Maximum Number Of Warewashing Cycles That Can
Produce A 5-Log Bacterial Reduction Of Escherichia coli K12 With A Single Batch Of
Detergent, Rinse Water And Sanitizer.
122
Whole Milk
3.6
4.8
4.7
Rep 1-1
AVERAGE
Rep 1-2
AVERAGE
Rep 2-1
AVERAGE
Rep 2-2
AVERAGE
Rep 3-1
AVERAGE
Rep 3-2
AVERAGE
Chocolate Milk 2% Milk
4.3
3.1
4.4
3.1
4.4
3.8
Skim Milk
2.8
2.6
2.6
4.367
4.367
3.333
2.667
5.2
3.9
4.8
4
3.7
3.9
3.2
3.5
3.1
2.4
2.5
2.2
4.633
3.867
3.267
2.367
4.4
4.8
4.3
3.8
4.5
4.1
3.4
3.4
3.5
2.2
2.1
2.5
4.5
4.133
3.433
2.267
4.3
4.8
5
3.8
3.7
3.7
3.4
3.5
3.3
2.3
2.4
2.4
4.7
3.733
3.4
2.367
4.4
4.8
4.7
3.5
3.4
3.9
3
3.1
3.1
2.7
2.9
2.7
4.633
3.6
3.067
2.767
4.8
4.9
4.7
4.5
4.5
4.2
3.4
3.3
3.1
2.8
2.4
2.6
4.8
4.4
3.267
2.6
Table A.3. Raw Data For The Force Required To Remove A 5 x 5 um Area Of Milk
Film From A Glass Surface After A Modified Washing Protocol With Sodium
Hypochlorite.
123
Rep 1-1
AVERAGE
Rep 1-2
AVERAGE
Rep 2-1
AVERAGE
Rep 2-2
AVERAGE
Rep 3-1
AVERAGE
Rep 3-2
AVERAGE
Whole Milk
5.1
4.9
4.7
Chocolate Milk
3.2
3.4
3.9
2% Milk
3.1
3
3
Skim Milk
2.2
2.4
2.2
4.9
3.5
3.033
2.267
4.9
4.8
4.5
4.1
4.2
4.1
3.1
3
2.9
2.5
2.6
2.4
4.733
4.133
3
2.5
4.5
4.5
4.6
3.4
3.7
3.8
3.2
3
3
2.8
2.4
2.4
4.533
3.633
3.067
2.533
4.3
4.8
4.7
3.4
3.8
3.6
2.9
2.7
2.8
2.1
2.4
2.5
4.6
3.6
2.8
2.333
4.7
4.5
4.5
4.2
4.3
4.3
2.9
2.7
2.7
2.9
2.8
2.8
4.567
4.267
2.767
2.833
4.8
4.9
4.2
4
4.1
4
2.5
2.9
3.1
2.4
2.3
2.3
4.633
4.033
2.833
2.333
Table A.4. Raw Data For The Force Required To Remove A 5 x 5 um Area Of Milk
Film From A Glass Surface After A Modified Washing Protocol With QAC.
124
Rep 1-1
AVERAGE
Rep 1-2
AVERAGE
Rep 2-1
AVERAGE
Rep 2-2
AVERAGE
Rep 3-1
AVERAGE
Rep 3-2
AVERAGE
Whole Milk
4.6
4.9
5.1
Chocolate Milk
4.3
4.4
4.4
2% Milk
3.1
3
3.2
Skim Milk
2.3
2.6
2.4
4.867
4.367
3.1
2.433
5.2
5.2
5
4.3
4.5
4.2
2.7
2.9
3.1
2.4
2.4
2.6
5.133
4.333
2.9
2.467
4.9
4.7
4.7
4
4.1
4.4
3.2
2.7
2.6
2.3
2.2
2.4
4.767
4.167
2.833
2.3
4.5
4.6
4.7
4.3
4.4
4.4
2.8
2.7
2.9
2.8
2.6
2.6
4.6
4.367
2.8
2.667
4.7
4.8
4.8
4.2
4.1
4.2
3
3
3.1
2.5
2.6
2.3
4.767
4.167
3.033
2.467
4.6
4.5
4.6
4.3
4.3
4.1
2.8
2.9
2.8
2.5
2.4
2.5
4.567
4.233
2.833
2.467
Table A.5. Raw Data For The Force Required To Remove A 5 x 5 um Area Of Milk
Film From A Glass Surface After A Modified Washing Protocol With EO Water.
125
Rep 1-1
AVERAGE
Rep 1-2
AVERAGE
Rep 2-1
AVERAGE
Rep 2-2
AVERAGE
Rep 3-1
AVERAGE
Rep 3-2
AVERAGE
Whole Milk
3.4
3.2
3.3
Chocolate Milk
3.1
3
3.2
2% Milk
2.4
2.3
2.8
Skim Milk
1.1
1.3
1.2
3.3
3.1
2.5
1.2
3.3
3.4
3.8
2.8
2.7
2.8
2.4
2.6
2.3
0.9
1.1
1.4
3.5
2.767
2.433
1.133
3.1
3.2
3.2
3
3.1
3.2
2.1
2.3
2.3
1.2
1.3
1.3
3.167
3.1
2.233
1.267
3
2.8
3.2
2.7
2.8
2.8
2.4
2.5
2.5
1.4
1.5
1.4
3
2.767
2.467
1.433
3.5
3.6
3.2
2.9
2.9
3.1
2.6
2.6
2.4
1.7
1.5
1.4
3.433
2.967
2.533
1.533
3.4
3.5
3.7
2.7
2.9
3
2.4
2.1
2.2
1.4
1.2
1.3
3.533
2.867
2.233
1.3
Table A.6. Raw Data For The Force Required To Remove A 5 x 5 um Area Of Milk
Film From A Glass Surface After A Modified Washing Protocol With PRO-SAN®.
126
Whole Milk
144.43
148.62
144.85
Rep 1-1
AVERAGE
Rep 1-2
AVERAGE
Rep 2-1
AVERAGE
Rep 2-2
AVERAGE
Rep 3-1
AVERAGE
Rep 3-2
AVERAGE
Chocolate Milk 2% Milk
77.123
56.647
117.99
73.431
122.86
49.431
Skim Milk
49.597
48
45.229
145.967
105.991
59.836
47.609
150.81
145.53
141.83
102.21
65.626
85.18
98.271
72.172
78.298
41.96
38.123
33.261
146.057
84.339
82.913
37.781
116.52
152.51
166.9
53.555
79.269
49.546
71.789
80.672
79.4
51.35
72.28
49.352
145.31
60.79
77.287
57.661
92.059
175.44
149.65
77.182
49.97
76.448
49.661
72.546
78.679
47.67
55.938
51.771
139.049
67.867
66.962
51.793
173.25
128.76
162.07
92.503
80.192
55.399
74.255
87.585
86.755
49.336
75.34
62.967
154.693
76.031
82.865
62.548
138.33
158.45
151.15
75.454
75.642
59.552
89.974
63.974
82.154
43.77
56.059
72.251
149.31
70.216
78.701
57.36
Table A.7. Raw Data Of Milk Film Thickness (nm) After A Modified Washing Protocol
With Sodium Hypochlorite.
127
Rep 1-1
AVERAGE
Rep 1-2
AVERAGE
Rep 2-1
AVERAGE
Rep 2-2
AVERAGE
Rep 3-1
AVERAGE
Rep 3-2
AVERAGE
Whole Milk
164.74
166.02
137.1
Chocolate Milk
95.209
85.729
73.984
2% Milk
64.088
72.332
70.841
Skim Milk
33.532
37.615
35.235
155.953
84.974
69.087
35.461
126.49
122.71
131.95
94.914
85.569
92.125
68.579
59.755
69.788
38.248
33.438
50.121
127.05
90.869
66.041
40.602
181.07
180.87
147.84
94.255
67.292
97.884
65.604
56.493
77.57
49.468
42.986
41.429
169.926
86.477
66.556
44.628
147.96
153.95
141.59
71.956
62.691
97.884
68.184
46.396
49.462
31.39
42.55
31.185
147.833
77.510
54.681
35.042
148.84
161.39
132.69
80.006
107.75
97.639
44.867
51.621
46.473
48.808
33.836
45.35
147.64
95.132
47.654
42.665
151.06
152.93
155.19
110.65
76.864
94.369
68.338
68.803
70.066
38.205
33.836
33.101
153.06
93.961
69.069
35.047
Table A.8. Raw Data Of Milk Film Thickness (nm) After A Modified Washing Protocol
With QAC.
128
Rep 1-1
AVERAGE
Rep 1-2
AVERAGE
Rep 2-1
AVERAGE
Rep 2-2
AVERAGE
Rep 3-1
AVERAGE
Rep 3-2
AVERAGE
Whole Milk
137.96
135.76
130.07
Chocolate Milk
117.29
104.59
122.96
2% Milk
50.511
36.294
57.724
Skim Milk
37.724
42.269
45.447
134.597
114.947
48.176
41.813
124.79
136.53
138.2
110.84
82.927
105.82
68.455
55.506
62.377
41.08
38.973
35.551
133.173
99.862
62.113
38.535
182.49
183.23
141.02
94.202
107.26
93.957
67.038
69.846
46.464
45.645
56.408
52.474
168.913
98.473
61.116
51.509
136.38
145.45
153.45
75.035
80.943
104.99
49.351
60.782
45.322
53.515
41.942
59.02
145.093
86.989
51.818
51.492
146.93
173.59
157.44
99.714
94.364
109.87
45.496
79.199
57.326
41.398
40.942
50.637
159.32
101.316
60.674
44.326
124.4
143.81
151.07
95.577
91.414
105.9
54.626
61.386
71.96
33.156
38.462
55.929
139.76
97.630
62.657
42.516
Table A.9. Raw Data Of Milk Film Thickness (nm) After A Modified Washing Protocol
With EO-Water.
129
Rep 1-1
AVERAGE
Rep 1-2
AVERAGE
Rep 2-1
AVERAGE
Rep 2-2
AVERAGE
Rep 3-1
AVERAGE
Rep 3-2
AVERAGE
Whole Milk
147.35
127.07
137.02
Chocolate Milk
70.17
64.715
61.921
2% Milk
42.956
46.593
38.077
Skim Milk
20.436
27.663
22.319
137.147
65.602
42.542
23.473
138.32
112.36
114.19
55.883
50.372
53.535
46.328
50.213
47.94
13.989
10.813
17.038
121.623
53.263
48.160
13.947
102.63
112.7
106.83
67.199
62.833
57.988
25.813
45.927
41.489
16.172
15.033
14.747
107.387
62.673
37.743
15.317
102.07
87.226
90.471
69.646
78.789
83.061
36.493
49.491
39.299
13.82
20.141
32.904
93.256
77.165
41.761
22.288
103.17
108.43
102.89
79.37
87.185
73.484
40.327
49.241
55.869
15.625
20.849
12.338
104.83
80.013
48.479
16.271
115.34
111.27
117.95
87.611
80.062
97.766
28.755
36.071
38.801
19.59
20.105
25.326
114.853
88.479
34.542
21.674
Table A.10. Raw Data Of Milk Film Thickness (nm) After A Modified Washing
Protocol With PRO-SAN®.
130
APPENDIX B: STATISTICAL ANALYSIS
131
Type III Sum of
Source
Squares
Df
Mean Square
F
Sig.
Corrected Model
2050.021a
7
292.860
312.861
.000
Intercept
20187.674
1
20187.674
21566.408
.000
55.854
3
18.618
19.890
.000
1863.931
2
931.965
995.615
.000
126.562
1
126.562
135.206
.000
3.674
1
3.674
3.925
.050
Error
127.306
136
.936
Total
22365.000
144
2177.326
143
Treatment
Dishware
Type
Bacteria
Corrected Total
Table B.1. Multifactorial ANOVA For Sanitizers Data For The Washing Experiment.
(I)
(J)
Treatment
Treatment
Tukey HSD NEW
Pro-San
Chlorine
(I-J)
Std. Error
Sig.
Lower Bound
Upper Bound
Pro-San
-.72*
.228
.010
-1.32
-.13
Chlorine
.94*
.228
.000
.35
1.54
QAC
.53
.228
.100
-.07
1.12
NEW
.72*
.228
.010
.13
1.32
Chlorine
1.67*
.228
.000
1.07
2.26
QAC
1.25*
.228
.000
.66
1.84
NEW
-.94
*
.228
.000
-1.54
-.35
-1.67*
.228
.000
-2.26
-1.07
QAC
-.42
.228
.265
-1.01
.18
NEW
-.53
.228
.100
-1.12
.07
Pro-San
-1.25*
.228
.000
-1.84
-.66
Chlorine
.42
.228
.265
-.18
1.01
Pro-San
QAC
95% Confidence Interval
Mean Difference
Table B.2. Multiple Comparisons (Tukey HSD and Bonferroni) For Various Sanitizers
Used In The Washing Experiment (continued).
132
Table B.2.: (continued)
Bonferroni
NEW
Pro-San
Chlorine
Pro-San
-.72*
.228
.011
-1.33
-.11
Chlorine
.94*
.228
.000
.33
1.56
QAC
.53
.228
.133
-.08
1.14
NEW
.72*
.228
.011
.11
1.33
Chlorine
1.67*
.228
.000
1.06
2.28
QAC
1.25*
.228
.000
.64
1.86
NEW
-.94*
.228
.000
-1.56
-.33
-1.67*
.228
.000
-2.28
-1.06
QAC
-.42
.228
.419
-1.03
.19
NEW
-.53
.228
.133
-1.14
.08
Pro-San
-1.25*
.228
.000
-1.86
-.64
Chlorine
.42
.228
.419
-.19
1.03
Pro-San
QAC
(J)
95% Confidence Interval
Mean
(I) Dishware Dishware Difference (I-J) Std. Error
Tukey HSD Plate
Tray
Glass
Bonferroni
Plate
Tray
Glass
Sig.
Lower Bound
Upper Bound
Tray
2.31*
.197
.000
1.84
2.78
Glass
-6.21*
.197
.000
-6.68
-5.74
Plate
-2.31*
.197
.000
-2.78
-1.84
Glass
-8.52*
.197
.000
-8.99
-8.05
Plate
6.21
*
.197
.000
5.74
6.68
Tray
8.52*
.197
.000
8.05
8.99
Tray
2.31*
.197
.000
1.83
2.79
Glass
-6.21*
.197
.000
-6.69
-5.73
Plate
-2.31*
.197
.000
-2.79
-1.83
Glass
-8.52*
.197
.000
-9.00
-8.04
Plate
6.21*
.197
.000
5.73
6.69
Tray
8.52*
.197
.000
8.04
9.00
Table B.3. Multiple Comparisons (Tukey HSD and Bonferroni) For Various Tableware
Items Used In The Washing Experiment.
133
FOOD SAMPLE
Sanitizer
1 = Whole Milk
2 = 2 Percent
1 = Chlorine
2 = Quatenary
Ammonium
3 = Skim Milk
3 = PRO-SAN
4 = Chocolate Milk
4 = EO Water
Table B.4. Legend Used For AFM Statistical Analyses
Multiple Comparisons
Dependent Variable:Thickness (nm)
95% Confidence Interval
Tukey HSD
(I) Food
(J) Food
Mean Difference
Sample
Sample
(I-J)
1
2
80.0154*
2.45813
.000
73.6624
86.3683
3
100.4353*
2.45813
.000
94.0823
106.7882
4
55.4587*
2.45813
.000
49.1057
61.8117
1
-80.0154*
2.45813
.000
-86.3683
-73.6624
3
20.4199*
2.45813
.000
14.0669
26.7729
4
-24.5567*
2.45813
.000
-30.9096
-18.2037
1
-100.4353*
2.45813
.000
-106.7882
-94.0823
2
-20.4199*
2.45813
.000
-26.7729
-14.0669
4
-44.9766*
2.45813
.000
-51.3295
-38.6236
1
-55.4587*
2.45813
.000
-61.8117
-49.1057
2
24.5567*
2.45813
.000
18.2037
30.9096
3
44.9766*
2.45813
.000
38.6236
51.3295
2
3
4
Std. Error
Sig.
Lower
Upper
Bound
Bound
Table B.5. Multiple Comparisons (Tukey HSD and Bonferroni) For Film Thickness Of
Various Milk Samples (continued).
134
Table B.5: (continued)
Bonferroni
1
2
80.0154*
2.45813
.000
73.4839
86.5468
3
*
2.45813
.000
93.9039
106.9667
*
2.45813
.000
48.9273
61.9901
*
2.45813
.000
-86.5468
-73.4839
*
2.45813
.000
13.8885
26.9513
*
2.45813
.000
-31.0881
-18.0252
*
2.45813
.000
-106.9667
-93.9039
*
2.45813
.000
-26.9513
-13.8885
*
2.45813
.000
-51.5080
-38.4451
*
2.45813
.000
-61.9901
-48.9273
*
2.45813
.000
18.0252
31.0881
*
2.45813
.000
38.4451
51.5080
4
2
1
3
4
3
1
2
4
4
1
2
3
100.4353
55.4587
-80.0154
20.4199
-24.5567
-100.4353
-20.4199
-44.9766
-55.4587
24.5567
44.9766
135
Mean
(I)
(J)
Sanitizers Sanitizers
Tukey HSD 1
2
3
4
Bonferroni
1
2
3
4
95% Confidence Interval
Difference
(I-J)
Std. Error
Sig.
Lower Bound
Upper Bound
2
3.4082
2.45813
.509
-2.9448
9.7612
3
26.5186*
2.45813
.000
20.1657
32.8716
4
.5049
2.45813
.997
-5.8481
6.8578
1
-3.4082
2.45813
.509
-9.7612
2.9448
3
23.1104*
2.45813
.000
16.7575
29.4634
4
-2.9033
2.45813
.639
-9.2563
3.4496
1
-26.5186
*
2.45813
.000
-32.8716
-20.1657
2
-23.1104*
2.45813
.000
-29.4634
-16.7575
4
-26.0137*
2.45813
.000
-32.3667
-19.6608
1
-.5049
2.45813
.997
-6.8578
5.8481
2
2.9033
2.45813
.639
-3.4496
9.2563
3
26.0137*
2.45813
.000
19.6608
32.3667
2
3.4082
2.45813
1.000
-3.1232
9.9396
3
26.5186*
2.45813
.000
19.9872
33.0500
4
.5049
2.45813
1.000
-6.0266
7.0363
1
-3.4082
2.45813
1.000
-9.9396
3.1232
3
23.1104*
2.45813
.000
16.5790
29.6418
4
-2.9033
2.45813
1.000
-9.4347
3.6281
1
-26.5186
*
2.45813
.000
-33.0500
-19.9872
2
-23.1104*
2.45813
.000
-29.6418
-16.5790
4
-26.0137*
2.45813
.000
-32.5452
-19.4823
1
-.5049
2.45813
1.000
-7.0363
6.0266
2
2.9033
2.45813
1.000
-3.6281
9.4347
3
26.0137*
2.45813
.000
19.4823
32.5452
Table B.6. Multiple Comparisons (Tukey HSD and Bonferroni) For Various Sanitizers
Used In AFM Analyses
136
Mean
(I)
(J)
Sanitizers Sanitizers
Tukey HSD 1
2
3
4
Bonferroni
1
2
3
4
95% Confidence Interval
Difference
(I-J)
Std. Error
Sig.
Lower Bound
Upper Bound
2
-3.5128
5.86349
.932
-18.9556
11.9300
3
33.5484*
5.86349
.000
18.1057
48.9912
4
-.0784
5.86349
1.000
-15.5212
15.3644
1
3.5128
5.86349
.932
-11.9300
18.9556
3
37.0613*
5.86349
.000
21.6185
52.5041
4
3.4344
5.86349
.936
-12.0083
18.8772
1
-33.5484*
5.86349
.000
-48.9912
-18.1057
2
-37.0613*
5.86349
.000
-52.5041
-21.6185
4
-33.6268*
5.86349
.000
-49.0696
-18.1840
1
.0784
5.86349
1.000
-15.3644
15.5212
2
-3.4344
5.86349
.936
-18.8772
12.0083
3
33.6268*
5.86349
.000
18.1840
49.0696
2
-3.5128
5.86349
1.000
-19.4477
12.4220
3
33.5484*
5.86349
.000
17.6136
49.4833
4
-.0784
5.86349
1.000
-16.0132
15.8564
1
3.5128
5.86349
1.000
-12.4220
19.4477
3
37.0613*
5.86349
.000
21.1264
52.9961
4
3.4344
5.86349
1.000
-12.5004
19.3693
1
-33.5484*
5.86349
.000
-49.4833
-17.6136
2
-37.0613*
5.86349
.000
-52.9961
-21.1264
4
-33.6268*
5.86349
.000
-49.5617
-17.6920
1
.0784
5.86349
1.000
-15.8564
16.0132
2
-3.4344
5.86349
1.000
-19.3693
12.5004
3
33.6268*
5.86349
.000
17.6920
49.5617
Table B.7. Multiple Comparisons (Tukey HSD and Bonferroni) For Whole Milk-Film
Thickness For Various Sanitizers Used In AFM Analyses.
137
Mean
(I)
(J)
Sanitizers Sanitizers
Tukey HSD 1
2
3
4
Bonferroni
1
2
3
4
95% Confidence Interval
Difference
(I-J)
Std. Error
Sig.
Lower Bound
Upper Bound
2
12.5797*
3.58542
.004
3.1367
22.0226
3
32.5562*
3.58542
.000
23.1132
41.9991
4
17.0017*
3.58542
.000
7.5587
26.4447
1
-12.5797*
3.58542
.004
-22.0226
-3.1367
3
19.9765*
3.58542
.000
10.5335
29.4195
4
4.4221
3.58542
.608
-5.0209
13.8650
1
-32.5562*
3.58542
.000
-41.9991
-23.1132
2
-19.9765*
3.58542
.000
-29.4195
-10.5335
4
-15.5544*
3.58542
.000
-24.9974
-6.1115
1
-17.0017*
3.58542
.000
-26.4447
-7.5587
2
-4.4221
3.58542
.608
-13.8650
5.0209
3
15.5544*
3.58542
.000
6.1115
24.9974
2
12.5797*
3.58542
.005
2.8358
22.3235
3
32.5562*
3.58542
.000
22.8123
42.3000
4
17.0017*
3.58542
.000
7.2579
26.7456
1
-12.5797*
3.58542
.005
-22.3235
-2.8358
3
19.9765*
3.58542
.000
10.2326
29.7204
4
4.4221
3.58542
1.000
-5.3218
14.1659
1
-32.5562*
3.58542
.000
-42.3000
-22.8123
2
-19.9765*
3.58542
.000
-29.7204
-10.2326
4
-15.5544*
3.58542
.000
-25.2983
-5.8106
1
-17.0017*
3.58542
.000
-26.7456
-7.2579
2
-4.4221
3.58542
1.000
-14.1659
5.3218
3
15.5544*
3.58542
.000
5.8106
25.2983
Table B.8. Multiple Comparisons (Tukey HSD and Bonferroni) For 2 Percent Milk-Film
Thickness For Various Sanitizers Used In AFM Analyses.
138
Mean
(I)
(J)
Sanitizers Sanitizers
Tukey HSD 1
2
3
4
Bonferroni
1
2
3
4
95% Confidence Interval
Difference
(I-J)
Std. Error
Sig.
Lower Bound
Upper Bound
2
13.3622*
3.23490
.001
4.8359
21.8884
3
30.3890*
3.23490
.000
21.8627
38.9153
4
5.3279
3.33446
.387
-3.4607
14.1166
1
-13.3622*
3.23490
.001
-21.8884
-4.8359
3
17.0268*
3.23490
.000
8.5006
25.5531
4
-8.0342
3.33446
.085
-16.8229
.7544
1
-30.3890*
3.23490
.000
-38.9153
-21.8627
2
-17.0268*
3.23490
.000
-25.5531
-8.5006
4
-25.0611*
3.33446
.000
-33.8497
-16.2724
1
-5.3279
3.33446
.387
-14.1166
3.4607
2
8.0342
3.33446
.085
-.7544
16.8229
3
25.0611*
3.33446
.000
16.2724
33.8497
2
13.3622*
3.23490
.001
4.5629
22.1614
3
30.3890*
3.23490
.000
21.5897
39.1883
4
5.3279
3.33446
.689
-3.7422
14.3980
1
-13.3622*
3.23490
.001
-22.1614
-4.5629
3
17.0268*
3.23490
.000
8.2276
25.8261
4
-8.0342
3.33446
.113
-17.1043
1.0358
1
-30.3890*
3.23490
.000
-39.1883
-21.5897
2
-17.0268*
3.23490
.000
-25.8261
-8.2276
4
-25.0611*
3.33446
.000
-34.1312
-15.9910
1
-5.3279
3.33446
.689
-14.3980
3.7422
2
8.0342
3.33446
.113
-1.0358
17.1043
3
25.0611*
3.33446
.000
15.9910
34.1312
Table B.9. Multiple Comparisons (Tukey HSD and Bonferroni) For Skim Milk-Film
Thickness For Various Sanitizers Used In AFM Analyses.
139
Mean
(I)
(J)
Sanitizers Sanitizers
Tukey HSD 1
2
3
4
Bonferroni
1
2
3
4
95% Confidence Interval
Difference
(I-J)
Std. Error
Sig.
Lower Bound
Upper Bound
2
-8.9852
5.21750
.320
-22.7266
4.7562
3
6.3395
5.21750
.620
-7.4019
20.0809
4
-22.3307*
5.21750
.000
-36.0721
-8.5893
1
8.9852
5.21750
.320
-4.7562
22.7266
3
15.3247*
5.21750
.023
1.5833
29.0661
4
-13.3454
5.21750
.060
-27.0869
.3960
1
-6.3395
5.21750
.620
-20.0809
7.4019
2
-15.3247*
5.21750
.023
-29.0661
-1.5833
4
-28.6702*
5.21750
.000
-42.4116
-14.9288
1
22.3307*
5.21750
.000
8.5893
36.0721
2
13.3454
5.21750
.060
-.3960
27.0869
3
28.6702*
5.21750
.000
14.9288
42.4116
2
-8.9852
5.21750
.538
-23.1645
5.1940
3
6.3395
5.21750
1.000
-7.8397
20.5187
4
-22.3307*
5.21750
.000
-36.5099
-8.1514
1
8.9852
5.21750
.538
-5.1940
23.1645
3
15.3247*
5.21750
.027
1.1455
29.5040
4
-13.3454
5.21750
.077
-27.5247
.8338
1
-6.3395
5.21750
1.000
-20.5187
7.8397
2
-15.3247*
5.21750
.027
-29.5040
-1.1455
4
-28.6702*
5.21750
.000
-42.8494
-14.4909
1
22.3307*
5.21750
.000
8.1514
36.5099
2
13.3454
5.21750
.077
-.8338
27.5247
3
28.6702*
5.21750
.000
14.4909
42.8494
Table B.10. Multiple Comparisons (Tukey HSD and Bonferroni) For Chocolate MilkFilm Thickness For Various Sanitizers Used In AFM Analyses.
140
Mean
(I) Food
(J) Food
Difference
Sample
Sample
(I-J)
2
71.9703*
5.67231
.000
57.0310
86.9095
3
94.2725*
5.67231
.000
79.3332
109.2118
4
69.1921*
5.67231
.000
54.2529
84.1314
1
-71.9703*
5.67231
.000
-86.9095
-57.0310
3
22.3022*
5.67231
.001
7.3630
37.2415
4
-2.7782
5.67231
.961
-17.7174
12.1611
1
-94.2725*
5.67231
.000
-109.2118
-79.3332
2
-22.3022*
5.67231
.001
-37.2415
-7.3630
4
-25.0804*
5.67231
.000
-40.0196
-10.1411
1
-69.1921*
5.67231
.000
-84.1314
-54.2529
2
2.7782
5.67231
.961
-12.1611
17.7174
3
25.0804*
5.67231
.000
10.1411
40.0196
2
71.9703*
5.67231
.000
56.5550
87.3855
3
94.2725*
5.67231
.000
78.8572
109.6878
4
69.1921*
5.67231
.000
53.7769
84.6074
1
-71.9703*
5.67231
.000
-87.3855
-56.5550
3
22.3022*
5.67231
.001
6.8870
37.7175
4
-2.7782
5.67231
1.000
-18.1934
12.6371
1
-94.2725*
5.67231
.000
-109.6878
-78.8572
2
-22.3022*
5.67231
.001
-37.7175
-6.8870
4
-25.0804*
5.67231
.000
-40.4956
-9.6651
1
-69.1921*
5.67231
.000
-84.6074
-53.7769
2
2.7782
5.67231
1.000
-12.6371
18.1934
3
25.0804*
5.67231
.000
9.6651
40.4956
Tukey HSD 1
2
3
4
Bonferroni
95% Confidence Interval
1
2
3
4
Std. Error
Sig.
Lower Bound
Upper Bound
Table B.11. Multiple Comparisons (Tukey HSD and Bonferroni) For Film Thickness
With Sodium Hypochlorite And Various Milk-Types.
141
Mean
(I) Food
(J) Food
Difference
Sample
Sample
(I-J)
2
88.0628*
4.16182
.000
77.1017
99.0238
3
111.3365*
4.16182
.000
100.3754
122.2976
4
63.7197*
4.16182
.000
52.7587
74.6808
1
-88.0628*
4.16182
.000
-99.0238
-77.1017
3
23.2737*
4.16182
.000
12.3127
34.2348
4
-24.3431*
4.16182
.000
-35.3041
-13.3820
1
-111.3365*
4.16182
.000
-122.2976
-100.3754
2
-23.2737*
4.16182
.000
-34.2348
-12.3127
4
-47.6168*
4.16182
.000
-58.5778
-36.6557
1
-63.7197*
4.16182
.000
-74.6808
-52.7587
2
24.3431*
4.16182
.000
13.3820
35.3041
3
47.6168*
4.16182
.000
36.6557
58.5778
2
88.0628*
4.16182
.000
76.7525
99.3731
3
111.3365*
4.16182
.000
100.0262
122.6468
4
63.7197*
4.16182
.000
52.4094
75.0300
1
-88.0628*
4.16182
.000
-99.3731
-76.7525
3
23.2737*
4.16182
.000
11.9634
34.5840
4
-24.3431*
4.16182
.000
-35.6534
-13.0328
1
-111.3365*
4.16182
.000
-122.6468
-100.0262
2
-23.2737*
4.16182
.000
-34.5840
-11.9634
4
-47.6168*
4.16182
.000
-58.9271
-36.3065
1
-63.7197*
4.16182
.000
-75.0300
-52.4094
2
24.3431*
4.16182
.000
13.0328
35.6534
3
47.6168*
4.16182
.000
36.3065
58.9271
Tukey HSD 1
2
3
4
Bonferroni
95% Confidence Interval
1
2
3
4
Std. Error
Sig.
Lower Bound
Upper Bound
Table B.12. Multiple Comparisons (Tukey HSD and Bonferroni) For Film Thickness
With QAC And Various Milk-Types.
142
Mean
(I) Food
(J) Food
Difference
Sample
Sample
(I-J)
2
70.9780*
3.78864
.000
60.9998
80.9562
3
94.3544*
3.78864
.000
84.3762
104.3326
4
41.9832*
3.78864
.000
32.0050
51.9614
1
-70.9780*
3.78864
.000
-80.9562
-60.9998
3
23.3764*
3.78864
.000
13.3982
33.3546
4
-28.9948*
3.78864
.000
-38.9730
-19.0166
1
-94.3544*
3.78864
.000
-104.3326
-84.3762
2
-23.3764*
3.78864
.000
-33.3546
-13.3982
4
-52.3712*
3.78864
.000
-62.3494
-42.3930
1
-41.9832*
3.78864
.000
-51.9614
-32.0050
2
28.9948*
3.78864
.000
19.0166
38.9730
3
52.3712*
3.78864
.000
42.3930
62.3494
2
70.9780*
3.78864
.000
60.6819
81.2741
3
94.3544*
3.78864
.000
84.0583
104.6505
4
41.9832*
3.78864
.000
31.6870
52.2793
1
-70.9780*
3.78864
.000
-81.2741
-60.6819
3
23.3764*
3.78864
.000
13.0803
33.6725
4
-28.9948*
3.78864
.000
-39.2910
-18.6987
1
-94.3544*
3.78864
.000
-104.6505
-84.0583
2
-23.3764*
3.78864
.000
-33.6725
-13.0803
4
-52.3712*
3.78864
.000
-62.6674
-42.0751
1
-41.9832*
3.78864
.000
-52.2793
-31.6870
2
28.9948*
3.78864
.000
18.6987
39.2910
3
52.3712*
3.78864
.000
42.0751
62.6674
Tukey HSD 1
2
3
4
Bonferroni
95% Confidence Interval
1
2
3
4
Std. Error
Sig.
Lower Bound
Upper Bound
Table B.13. Multiple Comparisons (Tukey HSD and Bonferroni) For Film Thickness
With PRO-SAN® And Various Milk-Types.
143
Mean
(I) Food
(J) Food
Difference
Sample
Sample
(I-J)
2
89.0504*
4.25633
.000
77.8404
100.2604
3
101.7777*
4.25633
.000
90.5677
112.9876
4
46.9398*
4.25633
.000
35.7299
58.1498
1
-89.0504*
4.25633
.000
-100.2604
-77.8404
3
12.7273*
4.25633
.020
1.5173
23.9372
4
-42.1106*
4.25633
.000
-53.3205
-30.9006
1
-101.7777*
4.25633
.000
-112.9876
-90.5677
2
-12.7273*
4.25633
.020
-23.9372
-1.5173
4
-54.8378*
4.25633
.000
-66.0478
-43.6279
1
-46.9398*
4.25633
.000
-58.1498
-35.7299
2
42.1106*
4.25633
.000
30.9006
53.3205
3
54.8378*
4.25633
.000
43.6279
66.0478
2
89.0504*
4.25633
.000
77.4833
100.6175
3
101.7777*
4.25633
.000
90.2105
113.3448
4
46.9398*
4.25633
.000
35.3727
58.5070
1
-89.0504*
4.25633
.000
-100.6175
-77.4833
3
12.7273*
4.25633
.023
1.1601
24.2944
4
-42.1106*
4.25633
.000
-53.6777
-30.5434
1
-101.7777*
4.25633
.000
-113.3448
-90.2105
2
-12.7273*
4.25633
.023
-24.2944
-1.1601
4
-54.8378*
4.25633
.000
-66.4050
-43.2707
1
-46.9398*
4.25633
.000
-58.5070
-35.3727
2
42.1106*
4.25633
.000
30.5434
53.6777
3
54.8378*
4.25633
.000
43.2707
66.4050
Tukey HSD 1
2
3
4
Bonferroni
95% Confidence Interval
1
2
3
4
Std. Error
Sig.
Lower Bound
Upper Bound
Table B.14. Multiple Comparisons (Tukey HSD and Bonferroni) For Film Thickness
With NEW And Various Milk-Types.
144