The efficacy of ware-washing protocols for removal of foodborne viruses from utensils in restaurants and food service establishments
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
Lizanel Feliciano, M.S.
Graduate Program in Food Science and Technology
The Ohio State University
2012
Dissertation Committee:
Dr. Melvin A. Pascall, Advisor
Dr. Jianrong Li
Dr. Hua Wang
Dr. Gerald S. Frankel
Copyrighted by
Lizanel Feliciano
2012
ABSTRACT
Food contact surfaces may present a potential health hazard if they are not properly cleaned and sanitized. Contaminated surfaces (e.g. utensils, cutting boards, equipment) have been identified as sources of cross-contamination for food during preparation and when being served to consumers. Therefore, ensuring effective cleaning and sanitization of food contact surfaces may help in the spread of foodborne pathogens and incidence of outbreaks.
The FDA Food Code and NSF International standards mandate that all surfaces/tableware items should be free of food soils and that a minimum microbial reduction of 5 logs must be obtained before surface sanitization could be considered effective. To comply with these standards, food service establishments must clean and sanitize food contact surfaces either manually or mechanically. Unfortunately, the standards set for these ware-washing methods specifically address the reduction of bacterial numbers from food contact surfaces, and not viruses. Therefore, information regarding the effectiveness of these standards against viruses needs to be elucidated. The first part of this dissertation (Chapter 2) compared the efficacy of sodium hypochlorite
(chlorine) and quaternary ammonium compound (QAC) in reducing bacterial populations
(Escherichia coli K-12 and Listeria innocua) and murine norovirus (MNV-1) counts on different food contact surfaces (ceramic plates, stainless steel forks and drinking glasses).
Each microorganism was separately inoculated into 2% reduced fat UHT milk and cream ii cheese spread. The milk was used to contaminate the drinking glasses and the spreadable cream cheese was used on the ceramic plates and forks. All tableware items were manually and mechanically washed and sanitized. Bacterial and viral counts were then determined on the surface of each tableware item using the plaque assay and plate count methods, respectively.
This study found that QAC and sodium hypochlorite sanitizers had the ability to produce ≥ 5 log reductions on both E. coli and L. innocua in manual and mechanical ware-washing operations. However, they were unable to produce the same level of antiviral activity (≤ 3 logs) under similar conditions irrespective of the nature of the tableware item and the ware-washing protocol.
The second part of this dissertation (Chapter 3) evaluated the efficacy of the manual and mechanical ware-washing protocols to remove caliciviruses from food contact surfaces (ceramic plates, stainless steel forks and drinking glasses). Porcine sapovirus (PoSaV) was used as a surrogate for both noroviruses (NoVs) and sapoviruses
(SaVs). The tableware items were contaminated with the milk (drinking glasses) and cream cheese spread (ceramic plates and forks) inoculated with PoSaV. These were manually and mechanically washed and sanitized with different sanitizing solutions
(chlorine and QAC). Tap water was used as the control sanitizing solution. After the ware-washing operations, the viral counts on the surfaces were determined by 50% tissue culture infective dose (TCID50). The chlorine sanitizer was able to reduce PoSaV by approximately 2 logs when exposed to higher temperatures during mechanical ware-
iii washing (49ºC vs. 43ºC during manual ware-washing). The viral reductions achieved with the other sanitizers (QAC and control) were not significant (< 1 log).
The third part of this dissertation (Chapter 4) investigated the effect of different sanitizers [chlorine-based sanitizers (bleach and Chlor-Clean), QAC and tap water] on their abilities to remove milk samples from underlying ceramic and glass surfaces. Three types of milk samples were tested in this study: 1) plain milk; 2) milk inoculated with
MNV-1; and 3) milk inoculated with PoSaV. Atomic force microscopy (AFM) was used to determine the thicknesses of the milk films left after the surfaces were mechanically washed and sanitized. Results from this study suggested that milk samples contaminated with viruses tend to adhere to a greater extent (thicker films) than non-contaminated milk and that common sanitizing solutions (chlorine-based and QAC sanitizers) appeared not to effectively remove milk-virus deposits from simulated food contact surfaces.
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DEDICATION
To my family and my friend Edgardo J. Díaz Polo, who lost his battle against cancer.
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ACKNOWLEDGMENTS
I want to start thanking God for all His help and love all this time. I would like to thank my advisor Dr. Melvin Pascall for his dedication, support and guidance during these past years. I would also like to thank Dr. Jianrong Li, Dr. Hua Wang and Dr. Gerald
Frankel for being part of my committee members. Dr. Jaesung Lee, special thanks to you for helping me during many difficult times.
I would like to acknowledge Paul Courtright for his assistance and willingness to help at all times. Thanks Paul, you are the best! I would also like to acknowledge Joel
Hipp and Tom Gruesser from Hobart Corporation for their technical assistance during this research project. Also, I would like to acknowledge the National Center for Research
Resources for their financial support (Award Number UL1RR025755). Finally, I want to thank my family and friends for their unconditional love, support and prayers during this learning and challenging experience.
vi
VITA
May 2002 ...... Eduardo García Carrillo High School
2002-2007 ...... B.S. Animal Industry, University of Puerto
Rico- Mayagüez Campus
2007-2009 ...... M.S. Food Science and Technology, The
Ohio State University
2009 to present ...... Graduate Research Associate, Department
of Food Science and Technology, The Ohio
State University
PUBLICATIONS
1. Feliciano, L, Lee, J and Pascall, MA. 2012. Transmission electron microscopic analysis showing structural changes to bacterial cells treated with electrolyzed water and an acidic sanitizer. Journal of Food Science, 77:M182-M187.
2. Pascall, MA, Feliciano Sanchez, L. 2010. Smart packaging technology as a food safety tool. Proceedings from the VI. International Packaging Congress. September 2010. Istanbul Turkey. The ASD Turkish Packaging Manufacturing Association. Istanbul, Turkey. Vol 1. p. 225-234.
3. Feliciano, L, Lee, J, Lopes, J and Pascall, MA. 2010. Efficacy of sanitized ice in reducing bacterial load on fish fillet and in the water collected from the melted ice. Journal of Food Science, 100:1M231-M239.
vii
4. Tournas, VH, Feliciano, L and Katsoudas, EJ. 2010. Evaluation of the petrifilm dry rehydratable film for the enumeration of yeasts and moulds in naturally contaminated foods. Journal of Food Safety, 30:506-514.
5. Gunawan, AM, Park, SK, Pleitner, JM, Feliciano, L, Grant, AL, and Gerrard, DE. 2007. Contractile protein content reflects myosin heavy-chain isoform gene expression. Journal of Animal Science, 85:1247-1256.
FIELDS OF STUDY
Major Field: Food Science and Technology
viii
TABLE OF CONTENTS
Page
ABSTRACT………………………………………………………………………...... ii
DEDICATION……………………………………………………………………….. v
ACKNOWLEDGEMENTS………………………………………………………… vi
VITA………………………………………………………………………………….. vii
LIST OF FIGURES………………………………………………………………….. xv
LIST OF TABLES…………………………………………………………………… xvii
1. LITERATURE REVIEW………………………………………………………… 1
1.1. Foodborne illnesses: Microorganisms of public health concern, their prevalence and challenges…………………………………………………. 1
1.2. Viruses: important foodborne illness agents…………………………………….. 6
1.2.1. Introduction to viruses………………………….………………………….. 7
1.2.2. Structure: non-enveloped and enveloped viruses………………………...... 8
1.3. Foodborne viruses of importance………………………………………………… 9
1.3.1. Hepatitis E virus ……..………………………………………………...... 9
1.3.2. Hepatitis A virus ……...………………………………………..……...... 10
1.3.3. Human Rotavirus ……...…………………………………………………... 11
1.3.4. Human Norovirus ……...………………………………………………….. 12
ix
1.3.4.1. Transmission routes…………………………………….…………. 14
1.3.4.2. NoV infection: replication and cell growth……………………….. 14
1.3.4.3. Murine Norovirus 1 (MNV-1) as a surrogate……………………... 16
1.3.5. Human Sapovirus (SaV)…………………………………………………………. 16
1.3.5.1. Transmission routes……………………………………………….. 17
1.3.5.2. SaV infection: replication and cell growth………………………… 18
1.3.5.3. Porcine Sapovirus as a surrogate………………………………….. 18
1.4. Foodborne illness risk factors associated with restaurants and food service establishments……………………………………………………… 19
1.4.1. Cleaning and sanitization of food contact surfaces………………………… 20
1.4.2. Surfaces…………………………………………………………………….. 20
1.4.3. Types of soils………………………………………………………………. 22
1.4.4. Cleaning agents…………………………………………………………….. 24
1.4.4.1. Surfactants………………………………………………………….. 25
1.4.4.1.1. Soaps……………………………………………………... 26
1.4.4.1.2. Detergents (synthetic detergents)……………………...... 27
1.4.4.2. Acid cleaning agents………………………………………………. 28
1.4.4.3. Alkaline cleaning agents…………………………………………... 28
1.4.5. Sanitizing agents…………………………………………………………... 30
1.4.5.1. Chlorine-based sanitizers………………………………………...... 32
1.4.5.2. Sodium hypochlorite………………………………………………. 34
1.4.5.3. Quaternary Ammonium Compounds (QAC)……………………… 34
1.4.5.4. Acidic sanitizers…………………………………………………… 36 x
1.4.5.5. Other sanitizing agents…………………………………………….. 37
1.4.5.5.1. Electrolyzed water……………………………………….. 37
1.4.5.5.2. Ozone……………………………………………………. 39
1.5. Ware-washing protocols for the control of foodborne pathogens and the Food Code……………………………………………………………….. 43
1.5.1. Manual versus mechanical ware-washing…………………………………. 45
1.6. Adhesion of soils to surfaces and testing methods………………………………. 46
1.6.1. Atomic Force Microscopy (AFM) for the analysis of surfaces…………………………………………………………………….. 49
1.6.2 Studies where AFM has been used to study the removal of food residues from food contact surfaces……………………………...... 54
1.7. References……………………………………………………………………...... 56
2. EFFICACY OF SODIUM HYPOCHLORITE AND QUATERNARY AMMONIUM COMPOUNDS DURING WARE- WASHING OPERATIONS FOR SANITIZATION OF TABLEWARE ITEMS CONTAMINATED WITH NOROVIRUS AND SELECTED BACTERIA……………………………………………………….……... 82
2.1. Abstract………………………………………………………………………….. 82
2.2. Introduction……………………………………………………………………… 83
2.3. Materials and Methods…………………………………………………………... 85
2.3.1 Cell culture and virus stock………………………………………………… 85
2.3.2. Preparation of bacterial cultures…………………………………………… 86
2.3.3. Food samples preparation and inoculation………………………………… 87
2.3.3.1. Ceramic plates……………………………………………………... 87
2.3.3.2. Forks and drinking glasses………………………………………… 87
2.3.4. Preparation of the detergents and sanitizing solutions…………………….. 88
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2.3.5. Mechanical ware-washing and sanitization of contaminated tableware items…………………………………………………………….. 89
2.3.6. Manual ware-washing and sanitization of contaminated tableware items…………………………………………………………….. 90
2.3.7. Viral sampling of the tableware surfaces: MNV-1 plaque assay………...... 92
2.3.8. Bacterial enumeration of the contaminated tableware surfaces……………. 93
2.3.9. Statistical analysis…………………………………………………...... 94
2.4. Results and Discussion…………………………………………………………… 95
2.4.1. Effect of air-drying on the reduction of MNV-1, E. coli and L. innocua on the contaminated tableware…………………………………. 95
2.4.2. Comparison between the efficacies of the mechanical and manual ware-washing protocols and the effect of the sanitizers……...... 96
2.5. Conclusions………………………………………………………………...... 105
2.6. References…………………………………………………………………...... 106
3. EFFICACY OF WARE-WASHING PROTOCOLS FOR THE REMOVAL OF CALICIVIRUSES FROM FOOD CONTACT SURFACES …………………………………………………………. 111
3.1. Abstract…………………………………………………………………………. 111
3.2. Introduction…………………………………………………………………….. 112
3.3. Materials and Methods…………………………………………………………. 114
3.3.1. Cell culture and virus stock………………………………………………. 114
3.3.2. Food samples preparation and inoculation……………………………….. 115
3.3.2.1. Ceramic plates…………………………………………………..... 115
3.3.2.2. Forks and drinking glasses……………………………………….. 116 xii
3.3.3. Preparation of the detergents and sanitizing solutions…………………… 116
3.3.4. Mechanical ware-washing and sanitization of contaminated tableware items……………………………………...... 117
3.3.5. Manual ware-washing and sanitization of contaminated tableware items…………………………………………………………... 118
3.3.6. Viral sampling of the tableware surfaces: PoSaV 50% infective dose assay……………………………………………………... 119
3.3.7. Statistical Analysis……………………………………………………..... 120
3.4. Results and Discussion………………………………………………………… 121
3.4.1. Effect of air-drying on the reduction of PoSaV on the contaminated tableware………………………………………………….. 121
3.4.2. Comparison between the efficacies of the mechanical and manual ware-washing protocols and the effect of the sanitizers tested………………………………………………………...... 122
3.5. Conclusions………………………………………………………...... 129
3.6. References……………………………………………………………………… 130
4. THE USE OF ATOMIC FORCE MICROSCOPY TO COMPARE THE EFFICACY OF DIFFERENT CHEMICAL SANITIZERS IN THE REMOVAL OF MILK CONTAMINATED WITH VIRUSES ON GLASS AND CERAMIC SURFACES……………………………………... 136
4.1. Abstract………………………………………………………………………… 136
4.2. Introduction……………………………………………………………………. 137
4.3. Materials and Methods………………………………………………...... 138
4.3.1. Food sample preparation and contact surfaces………………………...... 138
4.3.2. Mechanical ware-washing protocol…………………………………...... 140
4.3.3. Atomic force microscopy (AFM) measurements……………………….. 140
4.3.4. Statistical analysis………………………………………………………. 142 xiii
4.4. Results and Discussion………………………………………………………… 143
4.4.1. AFM measurements of the milk film thickness on ceramic and glass surfaces after mechanical ware-washing……………………… 143
4.5. Conclusion……………………………………………………………………... 151
4.6. References……………………………………………………………………… 153
5. CONCLUSION……………………………………………………………………. 156
6. REFERENCES……………………………………………………………………. 157
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LIST OF FIGURES
Figure 1.1. Sodium dodecyl sulfate (SDS) structure………………………………….. 26
Figure 1.2. General structure of quaternary ammonium compounds (QACs)………... 35
Figure 1.3. Schematic diagram of corona discharge ozone generator………………… 41
Figure 1.4. Schematic representation of a cold plasma ozone generator……………… 42
Figure 1.5. Contribution of the key factors to cleaning for hand and machine dishwashing………………………………………………………………... 46
Figure 1.6. Definition of contact angle (θ) from sessile drop geometry at solid/liquid/vapor triple point……………………………………………… 48
Figure 1.7. Schematic representation of the atomic force microscope………………… 52
Figure 1.8. Schematic of the scratch method with AFM tip on the surface of a sample…………………………………………………………. 53
Figure 2.1. Experimental design of the mechanical and manual ware-washing protocols followed for the removal of MNV-1, E. coli K-12 or L. innocua from different tableware items…………………………………. 91
Figure 2.2. Devices used during manual ware-washing to clean the different tableware items……………………………………………………………... 92
Figure 2.3. Survival of MNV-1, E. coli K-12 and L. innocua on different contaminated tableware before and after 1 hour air-drying………………... 96
Figure 2.4. Survival of MNV-1, E. coli K-12 and L. innocua on contaminated tableware items after washing and sanitizing, using the mechanical dishwasher………………………………………………………………….. 98
xv
Figure 2.5. Survival of MNV-1, E. coli K-12 and L. innocua on contaminated tableware items after washing and sanitizing, during manual ware-washing……………………………………………… 99
Figure 3.1. Survival of PoSaV on different contaminated tableware before and after 1 hour air-drying………………………………………… 122
Figure 3.2. Survival of PoSaV on contaminated tableware items after washing and sanitizing, using the mechanical and manual dishwasher, respectively…………………………………………………... 124
Figure 4.1. Illustration of milk samples on glass and ceramic surfaces inside the mechanical ware-washer……………………………………….. 139
Figure 4.2. A tapping 3-D topographical AFM image of the surface a milk-MNV-1 sample on the glass surface after chlorine sanitization and the contact mode scratching test………………………… 142
Figure 4.3. AFM mean thicknesses (nm) of milk samples on ceramic and glass surfaces…………………………………………………………. 146
Figure 4.4. Effect of the detergent solution on the film thickness left on bare ceramic and glass surfaces during the mechanical ware-washing protocol……………………………………………………. 146
xvi
LIST OF TABLES
Table 1.1. Common detergent ingredients……………………………………………… 28
xvii
CHAPTER 1: LITERATURE REVIEW
1.1. Foodborne illnesses: Microorganisms of public health concern, their prevalence
and challenges
Despite numerous food safety information campaigns, educational efforts and years of research in different microbiology disciplines, foodborne diseases are a significant cause of illness and death in the United States (Powell et al., 2011; Jacob et al., 2010; Griffith, 2006). According to the latest report from the Centers for Disease
Control and Prevention (CDC), each year approximately 48 million people (or 1 in 6
Americans) get sick, 128,000 are hospitalized, and 3,000 die of foodborne diseases
(CDC, 2011). Foodborne illness can be caused by different agents: chemical, physical or microbial agents. However, microbial agents (e.g. bacteria, parasites, viruses and prions) have gained special attention over the last two decades since they are the main cause of foodborne illness (Newell et al., 2010). It has been estimated that 31 known pathogens are responsible for 9.4 million episodes of foodborne illnesses in the US each year
(Scallan et al., 2011). The major pathogens associated with most of these illnesses, hospitalizations and deaths included Salmonella spp. (nontyphoidal), Clostridium perfringens, Campylobacter spp., Staphylococcus aureus, Toxoplasma gondii,
Escherichia coli (STEC) O157, Listeria monocytogenes and Norovirus (CDC, 2011;
1
Scallan et al., 2011). A brief description of these pathogens will be given in the following sections. However, more detailed information will be provided for viruses (section 1.2), including their prevalence in food, stability in the environment and concerns associated with their dissemination in food.
Salmonella spp.
Salmonella species are a leading bacterial cause of acute gastroenteritis. These are of great concern because of their ability to cause large outbreaks of foodborne illnesses involving a variety of foods of plant and animal origin (Holley, 2011). Nevertheless, foods of animal origin such as eggs, poultry, pork or beef, are generally associated with the transmission of this bacterial species (Pires et al., 2010; Callaway et al., 2008;
Braden, 2006). The global human health impact of non-typhoidal Salmonella is high, with an estimated 93.8 million illnesses, of which an estimated 80.3 million are foodborne, and 155,000 deaths each year (Majowicz et al., 2010). Non-typhoidal salmonellosis is commonly known to be a self-limiting enteric disease caused by different serovars of Salmonella enterica subspecies enterica, including serovar Enteridis and
Typhimurium (EFSA 2010; Tindall et al., 2005). In such outbreaks the problem is compounded by spread of the infection through person-to-person contact and the fact that many serovars exhibit multiple antibiotic resistances (Holley, 2011).
2
Clostridium perfringens
Clostridium perfringens is a spore forming bacterium that is the 2nd leading bacterial cause of foodborne illness in the US. It accounts for 10% of all foodborne illnesses (Scallan et al., 2011). This bacterium can cause food poisoning, gas gangrene
(clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal infections in humans. It is also a veterinary pathogen causing enteric diseases in both domestic and wild animals (Sawires and Songer, 2006; Van Immerseel et al., 2004).
Clostridium perfringens strains are divided into five different types (A-E), according to the major toxins they produce. However, only types A and C strains have been associated with human diseases (Lindström, et al., 2011).
Campylobacter spp.
Campylobacter causes large numbers of sporadic cases of illnesses, although the majority of the cases go undetected or underreported (Holley, 2011). It accounts for 9% of the foodborness illness in the US (Scallan et al., 2011). The most common mode of infecting humans is by the consumption of food or water that has been contaminated by animals’ feces or cross-contaminated after contact with other contaminated foods (Vally et al., 2009; Friedman et al., 2004; Kapperud et al., 2003; Neimann et al., 2003). Raw or undercooked poultry has been recognized as a major source of infection, but other sources, such as beef, pork, lamb, milk, water, and seafood, also have been associated with Campylobacter infections (Zhao et al., 2010; Lindmark et al., 2009; Levesque et al.,
2007). Campylobacter jejuni and Campylobacter coli are the two species commonly
3 known to cause diarrheal illness, although Campylobacter jejuni accounts for the majority of the campylobacteriosis cases (Wilson et al., 2009; Gillespie et al., 2002).
Individuals infected by Campylobacter jejuni could suffer complications that could result in septicemia and neuropathies such as Guillain-Barré syndrome (GBS), which affects 1 in1, 000 cases (Butzler, 2004).
Staphylococcus aureus
Staphylococcus aureus is a major human pathogen that causes a wide spectrum of infections, ranging from superficial wound infections to life-threatening septicemia and toxic-shock syndrome (Bore et al., 2007). Staphylococcal food poisoning is one of the most common foodborne diseases worldwide, and it results from the ingestion of staphylococcal enterotoxin. Enterotoxigenic strains of S. aureus are responsible for the pre-formation of these enterotoxins in food, especially those that are rich in protein content (Tang et al; 2011; Ostyn et al., 2010). This bacterium can be a resident skin pathogen, multiplying in moist, warm areas (e.g. the groin) and frequently residing in the nasopharynx, from where it contaminates the skin on a regular basis through hand contact
(Todd et al., 2008). S. aureus can be released into the environment through perspiration, aerosols from sneezing, and saliva onto food or food contact surfaces such as tableware
(Todd et al., 2008).
4
Toxoplasma goondi
The parasite Toxoplasma gondii causes a retinal infection that affects healthy and immunocompromised people in many countries (Furtado et al., 2011; Holland et al.,
1996) and sometimes can result in the death of immune-suppressed individuals (Contini,
2008). In addition, T. gondii can cause neurological problems in infected individuals
(Jones et al., 2009). In the US alone, it is estimated that 400-4,000 congenital infections
(Lopez et al., 2000) and up to 1.26 million cases of ocular disease occur each year
(Holland, 2003). Also, numerous cases of encephalitis and other systemic illnesses in immune-suppressed persons have been estimated (Jones et al., 2007). Infection with T. gondii can occur by different means: by ingestion of tissue cysts from undercooked meat, consuming food or drink contaminated with oocysts (from cat feces), or by accidentally ingesting oocysts from the environment (Dubey and Jones, 2008).
Shiga-toxin producing E. coli (STEC)
STEC O157 is another important pathogenic bacterium of public health concern.
STEC O157 frequently causes large outbreaks of severe enteric infections, including bloody diarrhea, hemorrhagic colitis (HC), and hemolytic-uremic syndrome (Tariq et al.,
2011). Hemolytic-uremic syndrome (HUS) is a major complication of STEC O157 infection, which occurs in approximately 6% of all laboratory confirmed cases, including
15% of patients < 5 years of age (Gould et al., 2009). Although young children and females are more susceptible to develop HUS after STEC O157 infection, elderly persons appear to have the highest proportion of deaths associated with STEC O157 infection
5
(Gould et al., 2009). In the US, E. coli O157:H7 (O157) is the most commonly identified serotype of STEC (CDC, 2009a). However, other STEC serogroups that express an O antigen other than O157 (non-O157 STEC) are common (Andreoli et al., 2002; Klein et al., 2002), and are associated with outbreaks (Brooks et al., 2005).
Listeria monocytogenes
Listeria monocytogenes is reported as a persistent life threatening bacterium. This pathogen is the causative agent of listeriosis in both animals and humans (Briers et al.,
2011; Kalekar et al., 2011). Most of the human listeriosis is foodborne, where ready-to- eat (RTE) food products such as meat, dairy, seafood, and fresh produce are more commonly associated with L. monocytogenes contamination during processing (Gilmour et al., 2010; Lianou and Sofos, 2007; Mead et al., 1999). It is estimated that in the US, L. monocytogenes alone accounts for 19 % of human deaths as a result of contaminated food being ingested (Scallan et al., 2011). Listeria can cause mild gastroenteritis or severe infections of the blood (septicemia) and/or the central nervous system, as well as abortion and stillbirth, depending on host susceptibility (Carpentier and Cerf, 2011). Strains of L. monocytogenes can be frequently found on surfaces in the food industry, particularly in refrigerated environments, despite the fact that these are routinely cleaned and disinfected
(Carpentier and Cerf, 2011).
6
1.2. Viruses: important foodborne illness agents
Many preventive efforts have been made to reduce infections caused by foodborne pathogens. Unfortunately, little progress has been accomplished in the last years (CDC, 2009b). Viruses have been recognized as the leading cause of foodborne illnesses. In the US, approximately 59% of foodborne illnesses are due to viruses and the rest due to bacteria (39%) and parasites (2%) (Scallan et al., 2011). Concerns regarding human exposure to viruses through contaminated water or food products are on the raise, since the number of documented foodborne virus outbreaks is believed to be on the increase globally. In addition to this, the increased consumption of foods traditionally eaten raw (e.g. fruits and vegetables) and globalization of international trade have increased the risks of viral contamination of foods (Hamburg, 2011; Gerba, 2006,
Koopmans et al., 2002). In view of this, more detailed information regarding foodborne viruses will be provided in the following sections and it will seek to provide an understanding of the unique features of viruses and how they persist in our food supply chain.
1.2.1. Introduction to viruses
Viruses are inert particles that need suitable host cells in order to replicate (Baert et al., 2007). Therefore, they are unable to multiply in food, water, or during storage by themselves (Li et al., 2012; Koopmans and Duizer, 2004). For this particular reason, the ability of contaminated food/water to serve as a vehicle of infection depends on the stability of the virus, the degree of initial contamination, the method of food/water
7 processing and storage, dose needed to produce infection, and susceptibility of the host
(Papafragkou et al., 2006; Koopmans et al., 2002). Viruses implicated in foodborne diseases are enteric in nature and are found in the human gut. They are excreted in human feces, and are transmitted by the fecal-oral route. Many different viruses are found in the human gut, but not all are recognized as food-borne pathogens (Greening, 2006). Foods that are minimally processed before consumption (fresh produce, raw mollusks and other ready-to-eat (RTE) foods) are the ones most commonly associated with foodborne viral infections (FAO/WHO, 2008). Foodborne viruses are fairly resistant to environmental stressors including pH, low temperatures, and some enzymes, particularly those found in the human gastrointestinal tract (Jaykus, 2000a). Therefore, it is not surprising that human enteric viruses are able to survive different food storage and processing conditions. This increases the likelihood that any kind of food product can potentially become a vehicle for foodborne virus transmission (Jaykus, 2000b).
1.2.2. Structure: non-enveloped and enveloped viruses
The structural composition of viruses plays an important role in their stability. In general, viruses are mainly comprised of two fundamental components: genetic material
(RNA or DNA) and a protein coat (capsid). Viral capsids have important functional properties including: 1) protection of the genetic material when the virion (infectious viral particle) is outside the host cell; and 2) the initiation of infection when the virion contacts a receptor on a suitable host cell (Cliver, 2009). The protection the capsid provides to the genetic material is vital and is a determinant factor in how the virus
8 ultimately infects its host cells. Virus infection only occurs if the viral genetic material is functional and if the capsid is able to attach to the receptor of its host’s cells in order to initiate the infection process (Cliver, 2009; Rodriguez et al., 2009).
Depending on their structural composition, viruses can be classified as enveloped or non-enveloped viruses. Enveloped viruses are those that contain a lipid membrane
(bilayer of phospholipids) acquired from their host cells during assembly (Chan et al.,
2010). The envelope adds not only a protective lipid membrane, but also an external layer of protein and sugars formed mainly by glycoproteins. Enveloped viruses attach to their host cell receptors by means of these viral membrane glycoproteins (Flint et al., 2004), therefore, these proteins play a major role in the viral life cycle. Non-enveloped viruses, on the other hand, lack this lipid membrane. Instead, they are composed of only a protein capsid and nucleic acid. These viruses then depend upon contact with an appropriate cell- surface receptor, or exposure to a specific intracellular environment in order to trigger conformational changes on their viral capsids, which ultimately lead to their host’s cells infection (Villanueva et al., 2005).
1.3. Foodborne viruses of importance
Viral infectious agents are of particular interest due to the potential health hazard they represent and the implications associated with the infections they cause. As mentioned earlier, the viruses associated with foodborne illnesses are enteric in nature and are typically transmitted by the fecal-oral route. These viruses include hepatitis E and
A, rotaviruses and caliciviruses (noroviruses and sapoviruses). The following sections
9 provide further information regarding the importance of these viruses as foodborne pathogens. However, more in depth discussion will be given on the caliciviruses, which are the main focus of this work.
1.3.1. Hepatitis E virus
Hepatitis E virus (HEV) is a small non-enveloped single-stranded RNA virus that belongs to the Hepeviridae family. This virus can be transmitted by four different routes:
1) drinking of contaminated water (waterborne transmission); 2) consuming raw or undercooked meat of infected wild animals (e.g. boars and deer) and domestic animals such as pigs (zoonotic foodborne transmission); 3) parenteral (bloodborne transmission), and vertical transmission from mother-to-child (perinatal transmission) (Mushahwar,
2008; Purcell and Emerson, 2001). It consists of four recognized major genotypes that infect humans and other animals: genotypes 1 and 2 are restricted to humans and are often associated with large outbreaks and epidemics in developing countries with poor sanitation conditions; genotypes 3 and 4 are zoonotic and infect humans and several other animals in both developing and industrialized countries (Meng, 2010). The unique features associated with these different HEV genotypes are of great importance as they correlate with the severity of the infections (Mizuo et al., 2005; Emerson and Purcell,
2003). For instance, it is believed that genotypes 3 and 4 are less pathogenic in humans, while genotype 1 has been shown to be more pathogenic (Navaneethan et al., 2008).
Therefore, the severity of the infection can differ from country to country, especially since epidemics have occurred only in the developing countries of Asia, Africa and
10
Mexico (Meng, 2010). Infections caused by HEV can be asymptomatic or they can induce clinical hepatitis, which may be severe or life threatening, particularly for pregnant women (Meng, 2010; Mushahwar, 2008).
1.3.2. Hepatitis A virus
Hepatitis A virus (HAV) is a small, non-enveloped, single-stranded RNA virus that is mainly transmitted via the fecal-oral route either by person-to-person contact or by contaminated water and food, particularly shellfish, fruits and salads (Sánchez et al.,
2007; Fiore, 2004). It is classified in the Picornaviridae family, although it differs in several important respects from most other human pathogens within this family (Feng and Lemon, 2010). The presence and severity of symptoms with hepatitis A virus infection is highly associated to the patient’s age. Approximately 70% of infected adults develop symptoms, including fever, malaise, anorexia, nausea, abdominal discomfort, and jaundice (Donnan et al., 2012). In children younger than 6 years of age, 70% of infections are asymptomatic. However, if illness does occur, it is typically not accompanied by jaundice (Fiore et al., 2006). In most developing countries, where HAV infection is endemic, the majority of persons are infected in early childhood, when the infection is generally asymptomatic. In developed countries, however, HAV infections are less common as a result of improved standards of living, but the majority of adults remain susceptible to infection by HAV, leading to a more severe disease outcome
(FAO/WHO, 2008).
11
1.3.3. Human Rotavirus
Rotavirus (RoV), a member of the Reoviridae family, is a large (70 nm), double- stranded, non-enveloped RNA virus (Weisberg, 2007). RoVs are transmitted by the fecal- oral route and cause disease in both humans and animals, especially domestic animals.
Even though RoV infections are not generally recognized as foodborne, outbreaks associated with food and water have been reported in a number of countries (Greening,
2006; Sattar et al., 2001). It is the most common cause of severe acute gastroenteritis in children in developing and in industrialized countries (Glass et al., 2006; Parashar et al.,
2003). It also represents an important cause of death in young children in low-income countries (EFSA, 2011; FAO/WHO, 2008). The health burden of this virus has led to the development of vaccines against this pathogen. Since 2006, RotaTeq and Rotarix have been licensed to be used as RoV vaccines. These are recommended for use in all countries by the WHO, particularly in those countries with high diarrhea-related mortality in children younger than 5 years of age (WHO, 2009a, 2009b and 2009c; Ruiz-Palacios et al., 2006; Vesikari et al., 2006).
1.3.4. Human Norovirus
Norovirus (NoV) is a small, non-enveloped, single-stranded RNA virus recognized as the most common cause of community-acquired diarrheal disease across all ages, and the most common cause of outbreaks of gastroenteritis and foodborne disease in the US (Scallan et al., 2011). It is responsible for at least 50% of all gastroenteritis outbreaks worldwide (Hall et al., 2011). Noroviruses are divided into five genogroups
12
(GI to GV). Genogroups I, II, and IV contain human pathogens and each genogroup is further divided into several genotypes (Zheng et al., 2006). However, NoVs belonging to the GII.4 genotype are the most prevalent, accounting for 85.8% of the global NoV foodborne outbreaks (Bull et al., 2006). Outbreaks associated with NoV occur in various settings, including hospitals, nursing homes, restaurants, childcare centers, and cruise ships (Lopman et al., 2012). Infection caused by NoV has an average incubation period between 24 and 48 h and the illness duration ranges between 12 and 60 h (Schwartz et al,
2011). Typical NoV infection symptoms include nausea, vomiting, diarrhea, and low- grade fever that last from 1 to 3 days (Sala et al., 2009). Although the majority of the
NoV infections are usually mild and complications are rare, the young, elderly and immune-compromised individuals are at risk for complications caused by severe diarrhea and vomiting (Rosenthal et al., 2010; Wilhelm et al., 2010; Roddie et al., 2009; Haustein et al., 2009).
This virus is highly contagious and only a few particles are sufficient to cause illness (Teunis et al., 2008; Estes et al., 2006; Duizer et al., 2004). In view of this potential threat, in 2009, the CDC and its state partners developed a national norovirus outbreak surveillance network (CaliciNet) to assist in linking multistate outbreaks to a common source (e.g. contaminated food) (Vega et al., 2011). Even though this surveillance network is a novel and valuable tool, there are still some things to consider.
For example, Hall et al. (2011) reported that since CaliciNet was developed recently, only
21 state and local health laboratories (as of January 2011) were reported as certified to submit norovirus sequences and epidemiologic outbreak data to the network (Hall et al.,
13
2011). In response to this, Yen et al. (2011) reported that more participation from other state and local health laboratories could cause CaliciNet to become a good system to monitor trends in norovirus outbreaks, modes of transmission, populations at risk and the best intervention strategies. Despite the benefits of CaliciNet, there is still the need to better understand features of NoV such as its structure, mode of infection, replication and persistence. This type of information will greatly assist in the prevention, control and perhaps elimination of health problems associated with NoV infections.
1.3.4.1. Transmission routes
Transmission of NoV most frequently occurs through direct contact with NoV shedders, or indirectly through environmental or food contamination with human feces or vomit, especially in closed settings such as nursing homes, hospitals, cruise ships, restaurants and hotels (Matthews et al., 2012; Hall et al., 2011; Verhoef et al., 2008). In the US, contaminated food has been previously recognized as the main vehicle of infection. Common foods associated with the transmission of norovirus include fresh produce, ready-to-eat foods, oysters, baked goods, and berries (Gibson and Schwab,
2011). However, recent reports have revealed that person-to-person transmission is associated to the majority of the norovirus outbreaks in the US (Yen et al., 2011;
Rosenthal et al., 2010; Doyle et al., 2009; Patel, et al., 2009). Also, it has been suggested that NoV transmission during outbreaks may involve other routes, including contaminated fomites, which may act as a reservoir of the virus and perpetuate the outbreaks (Lopman et al., 2012; Isakbaeva at al., 2005).
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1.3.4.2. NoV infection: replication and cell growth
Human histo-blood group antigens (HBGAs) appear to resemble the cell receptors to which NoVs attach to initiate infection (Cheetham et al., 2007; Huang et al., 2005;
Lindesmith et al., 2003). They are oligosaccharides, complex carbohydrates present on mucosal epithelial cell surfaces or as free antigens in blood, saliva, and other fluids
(Hansman et al., 2012; Esseili et al., 2012; Marionneau et al., 2001). NoVs belonging to the GII.4 genotype bind to more HBGAs than most other genotypes, and this helps to contribute to their worldwide transmission (Shirato et al., 2008). Even though viruses don’t grow in foods, since they are strict intracellular parasites and only replicate inside of host cells, they are very resistant and only a few virus particles are sufficient to cause illness (Teunis et al., 2008; Estes et al., 2006; Duizer et al., 2004). Therefore, the use of model systems that can lead to a greater understanding of the pathogenesis and the mechanisms NoVs use to remain infectious is necessary. In vivo propagation in human volunteers or in unsuspecting consumers of contaminated products is currently the only method to determine the infectivity of NoV (Richards, 2009). However, there are some factors to consider in human volunteers studies: 1) they require human subject protocols; and 2) it is uncommon and impractical to have people voluntarily become infected with a highly infectious and contagious agent that causes acute gastroenteritis (Li et al., 2012).
Consequently, cell culture systems currently used to study other viruses or infectivity assays in animal models are required. Nevertheless, NoVs are not only unable to propagate in current cell culture systems but they are unable to infect laboratory animals
(Richards, 2009; Koopmans and Duizer, 2004). Techniques such as immune-electron
15 microscopy, enzyme-linked immune-sorbent assays (ELISA) and radio-immunoassays have given way to more modern molecular biological techniques (reverse transcription- polymerase chain reaction (RT-PCR) based assays) to detect NoV. However, these methods are unable to differentiate infectious from inactivated viruses, thus limiting the practical application of molecular approaches for regulatory purposes (Richards, 2009).
1.3.4.3. Murine Norovirus 1 (MNV-1) as a surrogate
The fact that NoV lacks a cell culture system and there is no animal model for its study, has caused many researchers to turn to suitable surrogates such as feline calicivirus
(FCV) and MNV-1 (Baert et al., 2008; Cannon et al., 2006; Thurston-Enriquez et al.,
2005; Tree et al., 2005). MNV-1 is described as the ideal surrogate for NoV studies because of its genetic relatedness and its ability to survive under different stressors, including acidic conditions and high temperatures (Taube et al. 2009; Cannon et al. 2006;
Wobus et al. 2006). Also, MNV-1 is the only norovirus that replicates in cell culture and in a small animal model (Wobus et al., 2004; Karst et al., 2003). Even though MNV-1 shares common features with NoV, it also shares some differences. For example, MNV-1 does not cause the common symptoms of NoV infection (e.g. vomiting and diarrhea), although it can be found in the feces of infected mice, which are the small animal models used for MNV infectivity assays (Wobus et al., 2006; Karst et al., 2003). Also, MNV infections disseminate to multiple peripheral tissues and cause lethal systemic diseases
(Mumphrey et al., 2007). Despite these differences, the fact that MNV is the only
16 norovirus that infects a genetically defined host provides a unique opportunity for norovirus inactivation and control studies.
1.3.5. Human Sapovirus
Sapovirus (SaV) is another small, non-enveloped, single-stranded RNA virus that causes acute gastroenteritis in humans, particularly in children younger than 5 years old
(Chiba et al., 2000). It is not as well characterized as NoV, but is thought to be similar to
NoV in that it has a short incubation period (1-2 days), low infectious dose, causes a self- limiting illness that is rarely serious (with a significant percentage of asymptomatic infections) and is easily spread from person-to-person through fecal-oral transmission.
Both infections cause diarrhea, although a lesser percentage of SaV patients develop vomiting as compared to NoV patients (Rockx et al., 2002). Outbreaks of SaV infections have been reported in kindergartens, hospitals, mental healthcare facilities, hotels, cruise ships and restaurants (Dey et al., 2012; Yoshida et al., 2009; Blanton et al., 2006). Five genogroups of SaV (GI to GV), of which GI, GII, GIV and GV have been identified in humans (Dey et al., 2007). However, it has been suggested that diversity of SaVs observed in genogroups I and II could classify them into eight and five genotypes, respectively (Akihara et al., 2005).
1.3.5.1. Transmission routes
In comparison to NoVs, SaVs are rarely transmitted by contaminated food
(Buckow et al., 2008; Noel et al., 1997). However, recent studies suggest that sapovirus
17 may be transmitted from person-to-person by the fecal-oral route via contaminated food, water, and raw or undercooked shellfish, because its genome sequence has been detected in human feces (Iwakiri et al., 2009; Yoshida et al., 2009; Akihara et al., 2005), in untreated and treated river water (Haramoto et al., 2008; Hansman et al., 2007a), and clams consumed by humans (Hansman et al., 2008, 2007b). These reports show that there is conflicting opinions on food as a route of contamination for SaV. Hence, the reason why more research is needed on this issue. The speed with which SaVs can spread in close communities has led some investigators to hypothesize that airborne spread may also be an important infection route for these viruses, since the winter seasonality of SaV- associated disease could only be explained by an airborne spread (Dey et al., 2012).
1.3.5.2. SaV infection: replication and cell growth
Human SaV have neither been successfully cultured in vitro nor propagated in animal models. Therefore, SaV infection studies have been mostly conducted using traditional methods, such as electron microscopy (EM) and ELISA, which can detect intact virions and viral antigens, respectively (Pang et al., 2009; Wang et al., 2007).
Nowadays, the method most widely used is reverse transcription-PCR (RT-PCR), which has a high sensitivity and can also be used for genetic analysis since it detects viral RNA
(Hansman et al., 2007c; Wang et al., 2007). As with any virus, SaV needs a specific cell receptor in order to replicate. Unfortunately, a previous study conducted by Shirato-
Horikoshi et al., (2007) showed that SaV has no binding activity to HBGA (NoV cell receptors) or synthetic carbohydrates. Therefore, further studies are needed to identify
18
SaV cell receptors. Besides, since SaV studies have not been as extensively reported as
NoVs, there is little information regarding these viruses (Greening, 2006).
1.3.5.3. Porcine Sapovirus (PoSaV) as a surrogate
The Cowden strain of porcine enteric calicivirus (PEC) is the only member within the SaV genus that can be propagated in cell culture (Fullerton et al., 2007; Chang et al.,
2004; 2005). This virus can grow in a continuous porcine kidney cell line and requires the presence of bile acids in order to replicate (Chang et al., 2004; Parwani et al., 1991; Flynn and Saif, 1988). Also, the wild-type Cowden strain causes gastroenteritis in gnotobiotic pigs (Wang et al., 2012). The fact that this enteropathogenic calicivirus resembles gastroenteritis infections caused by NoVs and SaVs (Chiba et al., 2000; Flynn et al.,
1988), makes it an attractive surrogate that can be used to study these two viruses (NoV and SaV) (Wang et al., 2012).
1.4. Foodborne illness risk factors associated with restaurants and food service establishments
Restaurants and food service establishments are recognized as important sites for the transmission of foodborne illnesses (Verhoef et al., 2009; Goodgame, 2007; Hedberg et al., 2006). In a recent report, the Food and Drug Administration (FDA) mentioned the need for improvement in food handling practices as means of reducing foodborne illness outbreaks (FDA, 2009). This report documented observations collected from over 800 food establishments, and it revealed that out-of-compliance percentages remained high
19 for items related to: 1) improper food holding time and temperature; 2) poor employee personal hygiene; and 3) contaminated equipment. According to the documented observations, improper cleaning and sanitizing of food contact surfaces and utensils was the item most commonly found to be out-of-compliance. The report also showed that the percent of out-of-compliance observations for the cleaning and sanitizing of these items varied among food facility types. For example, the percent of out-of-compliance observations in fast-food restaurants was 41.7%, whereas in restaurants categorized as full-service, it was even higher (63.5%). Unfortunately, this high out-of-compliance percentage for the cleaning and sanitizing of food contact surfaces indicates that restaurants and food service establishments are in urgent need for improvement regarding this matter.
1.4.1. Cleaning and sanitization of food contact surfaces
The cleaning and sanitization of utensils and food contact surfaces (ware- washing) is designed to remove any visible food residues present on these surfaces. This is done in order to reduce or eliminate cross-contamination of “safe” foods when they get in contact with these “soiled” surfaces. The term “soil” is frequently used when referring to food debris or any unwanted matter on food contact surfaces (Verran et al., 2002). To optimize the ware-washing operations, different factors have to be taken into consideration. These include the nature of the food soil to be removed and the types of cleaning and sanitizing agents to be employed. To better understand the influence these factors may have during ware-washing operations in food service establishments or food
20 processing plants, the following sections will focus on the different types of surfaces to be cleaned, types of soils and some of the components and properties of different cleaning solutions.
1.4.2. Surfaces
Food contact surfaces need to be smooth, easy to clean, safe, durable, corrosion- resistant and sufficient in weight and thickness to withstand repeated ware-washing. This is so because the inability to effectively wash, rinse and sanitize these surfaces may lead to a buildup of pathogenic organisms that are transmissible through food (FDA Food
Code, 2009). Thus, to ensure the cleanability of food contact surfaces, it is important to understand that there are different types of surfaces (e.g. stainless steel, glass, ceramic) and each of them may have unique properties that can affect their soiling and cleanability. Some of these properties include hydrophobicity, porosity (roughness), polarity, surface area and charge (von Rybinski, 2007; Gould, 2003; Verran et al., 2000).
For instance, surface hydrophobicity seems to play an important role in the adhesion of microorganisms to surfaces. In fact, the adhesion of bacterial spores (Faille et al., 2002;
Hüsmark and Rönner 1993, 1992) and vegetative cells have been shown to increase with surface hydrophobicity (Sinde and Carballo 2000; Teixera and Oliveira 1999). In addition to this, the chances of getting surfaces with remnants of soils after cleaning may increase depending of the degree of surface irregularities (e.g. roughness, pits and crevices). This is so because soils situated in these irregularities are more strongly adhered to the surface due to higher contact area. Also, the number of attachment sites (related to surface area
21 and topography) on these surfaces will increase, resulting in stronger adhesion of the soils and enhanced protection from the mechanical and chemical action of the cleaning agents
(Detry et al., 2010; FDA Food Code, 2009; Jullien et al., 2002; Leclercq-Perlat and
Lalande, 1994). Polarity of surfaces has also shown to contribute to the adhesion of soils as well as microorganisms (Chen and Strevett., 2001; Flint et al., 2000; Boulange-
Petermann et al., 1993). An increase in surface polarity facilitates the spreadability or wettability of fluids or semi-solid foods on the surface. This wetting of the surface will only occur when the surface energy of the substrate surface is greater than that of the food soil (Saunders et al., 1992). Therefore, an increase in surface polarity causes an increase in molecular forces between surfaces and hence an increase in adhesion strength
(Awaja et al., 2009). This strong interaction between the surface and the food soil, as a result of an increase surface polarity, can then reduce the effectiveness of the cleaning and sanitizing agents used during ware-washing procedures (Whitehead et al., 2009).
More detailed information regarding the wettability concept is given in section 1.6.
1.4.3. Types of soils
Food soils can be categorized into four types, based on their major components: carbohydrates, fats, minerals and proteins (Jackson et al., 2008; Schmidt, 2009).
Carbohydrates are readily water-soluble, since they are rich in polar –OH groups
(Whitehead et al., 2009). Thus, any cleaning agent will be able to remove these deposits
(Sansebastiano et al., 2007). Fats, on the other hand, consist largely of non-polar hydrocarbons (Whitehead et al., 2009). Consequently they are water-insoluble and
22 difficult to remove. Both fats and oils are mixtures of triacylglycerols. They can be solids
(fats) or liquids (oils), depending on their fatty acid compositions and temperature
(Horton et al., 2006). In general, fats are solids at around 37ºC (body temperature) because they contain saturated long-chain fatty acyl (RC=O) groups (Horton et al., 2006).
As a result, high temperatures are generally used in cleaning processes to increase their fluidity and ease the action of cleaning chemicals (Detry et al., 2010). Mineral deposits
(inorganic) are formed when hardness salts are precipitated. They are insoluble in water and difficult to clean (Littlejohn and Grant, 2000). One major industry known for being affected by mineral deposition (i.e. milk-stone) on processing equipment is the dairy industry. Some of the minerals found in milk (e.g. calcium, magnesium, phosphate and citrate) are partly bound to proteins and partly free in solution. However, the equilibrium between bound and dissolved minerals can be changed, for instance, by changing the pH
(Jönsson and Trägårdh, 1990). For this reason, acid cleaning solutions are typically used in order for them to become soluble (Carsberg, 2003). Acid cleaning solutions increase the removal rates of minerals, which is attributed to an enhanced solubility and to the catalysis of hydrogen ions (Littlejohn et al., 1998). Protein deposits have strong adhesive properties and are structurally complex (Sansebastiano et al., 2007). It has been reported that when a protein attaches to an interface, it may undergo shape changes, the extent of which is dependent on the forces exerted by the surface (van der Veen et al. 2007). This behavior could then support the fact that proteins are able to decrease the hydrophilicity of polar substrates and to increase the polarity of hydrophobic substrates by adsorption
(Detry et al., 2010). Also, this may help to explain why protein deposits are the most
23 difficult to remove, requiring strong alkaline cleaning agents (Jackson et al., 2008). In addition to the previous statements, proteins have the tendency to denature and precipitate at high temperatures, which also affects their removal through cleaning procedures (Adhikary, 2006).
1.4.4. Cleaning agents
Cleaning agents are not intended to kill or inactivate pathogens, although they play a major role in reducing the total microbial load of contaminated surfaces (Holah,
2003). The primary functions of cleaning agents are: 1) to remove soil by decreasing the surface tension of water so that the soil can be dislodged or loosened; 2) to suspend soil particle in an emulsion by allowing the cleaning compound to surround the soil to form a micelle; and 3) to prevent re-suspension of the soil (Chmielewski and Frank, 2007).
Choosing the appropriate cleaning agents will help to ensure an effective sanitization of food contact surfaces during ware-washing operations. Inadequate cleaning procedures may result in enhanced adhesion of soils to substrate surfaces, and thus affecting the efficiency of the sanitizing solution to be subsequently applied (Carsberg, 2003). A good cleaning agent should meet the following criteria: 1) quick and complete solubility; 2) good wetting or penetrating action; 3) dissolving action on food solids; 4) emulsifying action on fat; 5) deflocculating, dispersing, or suspending action; 6) good rinsing properties; 7) complete water softening power; 8) non-corrosive on metal surfaces; 9) germicidal action; 10) economic in use (Stanfield, 2003).
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1.4.4.1. Surfactants
One major strategy in the proper cleaning of food contact surfaces is to use cleaning agents that are able to promote the wetting of surfaces and soils by loosening fats and other types of soils. To ensure the cleanibility of surfaces, cleaning agents generally contain surfactant agents. These are molecules that are incorporated into cleansing agents because of their ability to trap water-insoluble grease, fats and oils that are not effectively removed by water alone. In other words, surfactants help to remove water insoluble soils by surface wetting and then dispersal of them in the cleaning solution (Jackson et al., 2008). The molecular structure of surfactants is comprised of two groups: a hydrophilic (highly reactive with water) and hydrophobic group (not reactive with water). These two distinct groups are responsible for the cleaning action of surfactants, since the hydrophobic group binds to water insoluble soil materials whereas the hydrophilic group binds to water molecules and water soluble soils (Horton et al.,
2006; Friedman and Wolf, 1996). Figure 1.1 shows the chemical structure of sodium dodecyl sulfate (SDS), a common surfactant molecule. Surfactant agents function in soaps and synthetic detergents by promoting physical cleaning actions through emulsification, penetration, spreading, foaming and wetting (Schmidt, 2009).
25
Hydrophilic part
O CH3 CH2 CH2 CH2 CH2 CH2 - +
CH2 CH2 CH2 CH2 CH2 CH2 O S O Na
Hydrophobic part O
Figure 1.1. Sodium dodecyl sulfate (SDS), example of a surfactant molecule (Horton et al., 2006).
1.4.4.1.1. Soaps
Soaps are cleaning agents made from fatty acids or triglycerides (fats or oils) with alkali derivatives (e.g. sodium or potassium salts) through a process called saponification
(Cantarero et al., 2010; Kirsner and Froelich, 1998). These are commonly known as natural surfactants for the following reasons: 1) the fats and oils used for their production come from animal or plant sources; and 2) the cleansing action of soaps is mainly attributed to the presence of surfactant molecules (Abbas et al., 2004). Even though soaps offer several benefits, there are some negative aspects that limit their use as cleaning agents in food processing plants and food service operations. These include poor rinsing properties and the formation of insoluble residues (curds) when combined with hard water, due to the high number of electrolytes (e.g. magnesium and calcium) this water contains (Rosen and Kunjappu, 2012; Stanfield, 2003; Nix, 2000; Kirsner and Froelich,
1998).
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1.4.4.1.2. Detergents (synthetic detergents)
Detergents are widely used as alternatives to soaps. They are commonly known as surfactants and are surface active agents similar to soaps. Their primary function is to make water insoluble soils water-soluble so that they may be easily rinsed away (Horton et al., 2006). The term “detergent” includes any surfactant that has cleaning as well as emulsifying properties and as a result, it is often used as a general term to describe soaps and synthetic detergents (Kirsner and Froelich, 1998). Synthetic detergents are the most common cleaning compounds used today. Unlike soaps, they are not oil based and don’t form curds when exposed to hard water (Nagarajan and Sharma, 2001; Friedman and
Wolf, 1996). These cleaning agents contain a mixture of different compounds, each with a specific function (see Table 1.1). There are two classes of detergents: ionic and non- anionic. The ionic detergents include those that are anionic (negatively charged in water solution) and cationic (positively charged) in nature. Ionic detergents are generally characterized by their high foaming ability (form precipitates), which is usually not a desirable property. This is so because it is a common practice to blend detergent ingredients to optimize their cleaning properties. Ionic detergents tend to precipitate and do not blend well with other detergent ingredients (Schmidt, 2009). For this reason, nonionic (uncharged) detergents are preferred, since they have reduced foaming properties and don’t dissociate when dissolved in water (Schmidt, 2009). Nonionic detergents, then, are better solubilizing agents than ionics in very diluted solutions (Rosen and Kunjappu, 2012).
27
Table 1.1. Common detergent ingredients (Carsberg, 2003).
1.4.4.2. Acid cleaning agents
Acid cleaners are used most frequently to dissolve mineral deposits formed as a result of using alkaline detergents or that are present in hard water. Also, they are used to control and prevent mineral incrustation on surfaces. These are sometimes responsible for the corrosion of these surfaces and also promote bacterial biofilm formation (Salustiano et al., 2010). Acid cleaners include inorganic and organic acids. The common inorganic acids include phosphoric, nitric, sulfamic, sodium acid sulfate, and hydrochloric whereas the organic acids include hydroxyacetic, citric and gluconic (Schmidt, 2009). Organic 28 acids have the advantage of being less irritating and corrosive than inorganic acids (Le
Breton, 2009). Hence, they are more attractive for use as acid cleaners than inorganic acids.
1.4.4.3. Alkaline cleaning agents
Alkaline cleaners have a pH that ranges from 7 to 10. The higher the pH the stronger the action of the cleaner (Carsberg, 2003). Therefore, they are categorized according to their alkalinity strength: strongly, heavy-duty and mild alkaline cleaners.
Highly alkaline cleaners are best suited for use in areas with heavy and difficult-to- remove soils (e.g. proteins and fats), like those found in meat processing plants (Sharma et al., 2003). Proteins, for example, will hydrate and swell when they come into contact with water. This helps alkalis to react with them to form soluble salts (Parker, 2007).
These cleaners include sodium hydroxide (caustic soda) and silicates (Marriot, 2004).
Sodium hydroxide is preferred over other alkalines because of its ability to form a strong alkaline solution when dissolved in water (Madaeni et al., 2011). However, since it is highly corrosive and difficult to rinse, the addition of silicates and wetting agents are added to reduce the negative characteristics and improve their penetration and rinsing properties (Marriot, 2004; Chisti, 1999). Heavy-duty alkaline cleaners have less dissolving power than strongly alkaline cleaners, but are considered less corrosive. They are frequently used in mechanized systems, including clean-in-place (CIP) and high- pressure systems, and are considered most effective in removing fats from surfaces
(Sharma et al., 2003). Mild alkaline cleaners, on the other hand, are used for hand
29 cleaning lightly soiled areas, since the use of stronger alkaline cleaners can cause severe skin irritations (Marriot, 2004; Lavoué, 2003; Cowart et al., 2000). Other alkaline cleaners employed for the removal of heavy soils are sodium, potassium, or ammonium salts of phosphates or carbonates. Among these cleaners, carbonate-based cleaners are of limited use in food processing cleaning regimes because of their interaction with calcium and magnesium and their film forming characteristics (Schmidt, 2009).
1.4.5. Sanitizing agents
Once the surfaces are washed with a detergent and rinsed with water, the following step is the application of a sanitizer. The reason for this is to reduce pathogenic microorganisms which may be present on equipment and utensils even after the washing/rinsing (Arvanitoyannis and Kassaveti, 2009; McSwane et al., 2005). To achieve a proper sanitization, the sanitizing agent to be employed has to conform to the provisions given in the FDA Food Code. According to the Food Code (2009), sanitization is defined as 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 of representative disease microorganisms of public health importance. FDA regulations listed in the Code of Federal Regulations (21 CFR 178.1010) provide the different sanitizing solutions that may be safely used on food-processing equipment, utensils, and on other food-contact surfaces.
There are several factors affecting the action of chemical sanitizers on surfaces.
These include the concentration of the sanitizer, pH of the solution and the time of
30 exposure (Stanfield, 2003). Generally, the higher the concentration of a sanitizer, the faster and more effective is its action. However, chemical sanitizers that are too concentrated may be toxic if not used in accordance with the requirements listed in the
Code of Federal Regulations (CFR). Therefore, they must to be used according to the manufacturer’s instructions (for compliance with the Environmental Protection Agency regulations) and only for their intended purpose (Schmidt, 2009). FDA regulations provided in 40 CFR 180.940 lists concentrations of sanitizers that are considered safe
(Food Code, 2009). The efficacy of some sanitizers is pH dependent (Schmidt, 2009;
Gonzalez et al., 2004; Bremer et al., 2002). For example, chlorine-based sanitizers are more effective at lower pH (undissociated form), but as the pH rises, an increase in their degree of dissociation occur, lowering their effectiveness (see section 1.4.5.2). In contrast, quaternary ammonium compounds (QACs) generally have higher activity at alkaline pH. As the pH increases, the number of negatively charged groups on bacterial cell surface increases, thus enhancing their degree of binding (see section 1.4.5.3).
Additionally, the efficacy of sanitizing solutions may be enhanced by increasing their temperature. This is partly based on the principle that chemical reactions are generally faster as the temperature increases (Stanfield, 2003). However, temperatures that are greater than 55ºC are to be avoided, since most chemical sanitizers are corrosive in nature
(Schmidt, 2009). Likewise, the longer the time a sanitizing agent is in contact with a food contact surface, the more effective the action of the sanitizer (Food Code, 2009).
However, microorganism populations and bacterial cells may respond differently to the
31 action of the sanitizing solutions due to factors such as cell age, spore formation and other physiological factors (Stanfield, 2003).
Chemical sanitizing agents more frequently used in food processing environments include chlorine-based sanitizers, quaternary ammonium compounds, acid and alkaline sanitizers. These will be discussed in the following sections.
1.4.5.1. Chlorine-based sanitizers
Chlorine-based sanitizers are the most commonly used in the food industry. For example, produce processing facilities frequently use them to sanitize produce and surfaces as well as to reduce microbial populations in water used during cleaning and packing operations (Parish et al., 2003). Also, these sanitizers are commonly employed for the sanitization of equipments and utensils (Marriot, 2004). Chlorine-based sanitizers are recognized for their broad-spectrum properties and are believed to act on microbial membranes, inhibit cellular enzymes involved in glucose metabolism, have a lethal effect on DNA and oxidize cellular proteins (Schmidt, 2009). The killing action of these sanitizers is attributed to the formation of hypochlorous acid (HOCl) in solution, which is a form of free available chlorine that has the highest bactericidal activity (Parish et al.,
2003). The term “free available chlorine” is commonly used when the primary free forms of chlorine (HOCl and/or ClO-) are present in aqueous solution. However, when these free forms combine with ammonia and organic compounds, they are referred to as
“combined chlorine” (Gerba, 2009). The addition of chlorine to water produces HOCl
(Equation 1), which is most active and predominant from pH 4 to 7 (Fu et al., 2007).
32
+ - Cl2 + H2O → HOCl + H + Cl (Eq. 1)
Chlorine Water Hypochlorous Hydrogen Chloride acid ion ion
As the pH is increased (> 8), HOCl is converted to ClO- (Equation 2). The latter species is known for its poor antimicrobial activity (Fu et al., 2007; Estrela et al., 2002).
- - HOCl + OH → ClO + H2O (Eq. 2)
Hypochlorous Hydroxide Hypochlorite Water acid ion ion
Therefore, to maximize the killing action of these sanitizers, their pH should range between 4 and 7 because at lower pH it may sensitize the outer membrane of bacterial cells to the entry of HOCl (McPherson, 1993). In addition to this, the concentration of HOCl will be optimal and its dissociation minimized (Christensen et al.,
2008; McDonnell and Russell, 1999; Rutala and Weber, 1997). However, pH values between 6.0 and 7.5 are used in sanitizer solutions to minimize corrosion of the equipment while yielding acceptable chlorine efficacy (FDA, 2001).
Chlorine-based sanitizers are available in various forms including gaseous chlorine, chlorine dioxide, hypochlorous acid and hypochlorites (Wirtanen and Salo,
2003). However, hypochlorites (e.g. sodium and calcium hypochlorites) are more frequently used due to their economical and fast-acting properties (Marriot, 2004).
33
1.4.5.2. Sodium hypochlorite
Sodium hypoclorite (NaOCl) is one of the most frequently employed sanitizing agents in the food industry (Bagge-Ravn et al., 2003). When ready for use, the end-use concentration of all HOCl (free available chlorine) in the NaOCl solution is not to exceed
200 ppm on food contact surfaces (40 CFR 180.940). NaOCl is inexpensive, readily available and is produced by reacting chlorine gas with sodium hydroxide (Carsberg,
2003). There are several factors to take into consideration about these types of sanitizers.
For instance, the efficacy of chlorine is affected by pH, temperature of the treatment solution, amount of organic material other than the type of microorganism present, and concentration of free chlorine (Kreske et al., 2006; Cords et al., 2005). Also, hypochlorites not only cause skin irritation and mucous membrane damage, they can potentially interact with some chemicals and may lead to the formation of toxic chlorine gas (Rutala and Weber, 1997). Furthermore, NaOCl is highly corrosive to several metals, especially when employed at high temperatures (Schmidt, 2009).
1.4.5.3. Quaternary Ammonium Compounds
Quaternary ammonium compounds (QACs) are commonly used as sanitizers in the food industry. The most widely used in food-processing environments is benzyl ammonium chloride, also known as benzalkonium chloride (Cruz and Fletcher, 2012;
Mustapha and Liewen, 1989). QACs are mainly recognized for their effect on bacterial cells, although they have been shown to affect some viruses (enveloped), fungi, and protozoans (Fu et al., 2007; Russell et al., 1999). The selectivity of QAC has been
34 attributed to its ionic binding capabilities and hydrophobic interactions with microbial membrane surfaces. They are positively charged (see Figure 1.2) and when in contact with microorganisms their cationic heads are oriented outwards and their hydrophobic tails attracted to the lipid bilayers of the organisms. This causes rearrangement of the membranes and subsequently leakage of the intracellular constituents (Ioannou et al.,
2007; McBain et al., 2004; Russell and Chopra, 1996; Cabral, 1992; Gilbert and Al-Taae,
1985). Mixed-micelle aggregates with these hydrophobic membrane components are formed at QAC concentrations normally used for surface sanitization in the industry
(McBain et al., 2004; Salton, 1968). The reason for this is that they target carboxylic groups and cause general coagulation in the bacterial cytoplasm (To et al., 2002).
Figure 1.2. General structure of quaternary ammonium compounds (QACs) (modified from Schmidt, 2009).
In general, most of the QAC formulations do not require rinsing with water after being applied to surfaces (Buffet-Bataillon et al., 2012). Thus, they leave a residual
35 antimicrobial film on surfaces (Marriot, 2004), which prevents deposition and growth of bacterial biofilms (Kügler et al., 2005). QACs have several attributes that are not shared by other sanitizers: 1) they are stable to heat; 2) are effective over a wide pH range (but most effective in slightly alkaline solutions); 3) are non-corrosive and non-irritating; 4) are less affected by organic matter than chlorine; and 4) generally have no odor or taste
(Weddig et al., 2007). However, there are some specifications that are to be met when using these sanitizers, especially during sanitization of food-contact surfaces. For example, QAC solutions have to be used only in water with a hardness ≤ 500 ppm or if the hardness is not greater than what is specified by the EPA-registered label use instructions (Food Code, 2009). Also, their end-use concentration in solution is not to exceed 200 ppm (40 CFR 180.940).
1.4.5.4. Acidic sanitizers
Acid-based sanitizers are used widely in the food, dairy, and beverage industries to clean processing equipment of mineral deposits in addition to sanitizing surfaces (Fu et al., 2007). Acid-based sanitizers are capable of neutralizing excess alkalinity that remains after the application of cleaning compounds. Thus, they also prevent the formation of alkaline deposits and sanitize (Marriot, 2004). These negatively charged sanitizers are surface active agents and are effective against most bacteria, yeast and molds (Schmidt,
2009; Carsberg, 2003). Examples of these include organic acids such as acetic, peroxyacetic, lactic, propionic, and formic acid (Marriot, 2004). As is the case with
QACs, acid-based sanitizers can alter the permeability of bacterial cell membranes in
36 acidic environments (Fu, et al., 2007). The undissociated form of these acids is capable of penetrating the cell membrane lipid bilayer, dissociates (cell interior has a higher pH than the exterior) and consequently acidifies the cell interior. This leads to the death of the cell
(Davidson and Harrison, 2002). Some of the characteristics that make these sanitizers highly desirable include their stability, low pH use, low-corrosiveness and moderately affected activity of water hardness (Schmidt, 2009). Despite all the benefits these sanitizers offer, there are some factors to take into consideration including: 1) relatively high cost; 2) closely defined pH range of activity (2 to 3); and 3) excessive foaming in
CIP systems and incompatibility with cationic surfactants (Schmidt, 2009). Also, since they can lose their effectiveness in the presence of alkaline residuals or in the presence of cationic surfactants, all cleaning compounds should be rinsed from the intended surfaces before they are applied (Marriot, 2004).
1.4.5.5. Other sanitizing agents
Other types of sanitizing agents used in the food industry include trisodium phosphate, iodophores, hydrogen peroxide, electrolyzed water and ozone. The following sections will mainly focus on the sanitizing properties of electrolyzed water and ozone.
1.4.5.5.1. Electrolyzed water
Electrolyzed oxidizing (EO) water has been used in sectors such as agriculture, dentistry and medicine. It has also been used in the food industry to reduce or eliminate bacterial populations on food products, food-processing surfaces, and non-food contact
37 surfaces (Huang et al., 2008; Hricova et al., 2008). The electrolyzed water is produced by the electrolysis of a diluted salt solution (sodium chloride and tap water) in a chamber, where a semi-permeable membrane separates an anode from a cathode electrode (Hricova et al., 2008). Upon application of a current across the electrodes, two types of water are produced. The cathode produces basic EO water containing sodium hydroxide (pH >11) and having an oxidation reduction potential (ORP) of approximately -800 mV. The anode produces acidic EO water containing hypochlorous acid (pH < 3) and having an ORP >
1,000 mV (Huang et al., 2008; Fabrizio and Cutter, 2003; Kim et al., 2000). The fact that the antimicrobial activity and ORP of EO water increase when the pH decreases, favors the application of acidic EO water as a sanitizer over basic EO water. This basic EO water is more frequently used as a degreaser to remove dirt from various surfaces (Hsu,
2008; Huang et al., 2008). In addition to its low pH and high ORP, acidic EO water
- contains chlorine, which may be available in three forms, HOCl, OCl and Cl2 (Hricova et al., 2008). Of these three, HOCl has the most bactericidal effect (Huang et al., 2008;
Parish et al., 2003; Len et. al., 2000).
The antimicrobial activity of acidic EO water (AEW) is attributed to its low pH and this reduces bacterial growth and makes the bacterial cells more sensitive to active chlorine by sensitizing their outer membrane to the entry of HOCl (Park et al., 2004).
When HOCl penetrates cell membranes, it produces hydroxyl radicals which exert their antimicrobial activity by causing oxidation of key metabolic systems (Hricova et al.,
2008). Although the killing action of chlorine compounds has been mainly attributed to the destruction of cell membranes, other modes of action proposed including the
38 decarboxylation of amino acids, reactions with nucleic acids, and unbalanced metabolism after the destruction of key enzymes (Mahmoud, 2007; Mahmoud et al., 2004; Kiura et al., 2002; Koseki et al., 2000).
Even though there are advantages that make acidic AEW an attractive sanitizer, concerns with its use include chlorine gas emission, metal corrosion, and synthetic resin degradation due to its strong acidity and free chlorine content (Guentzel et al., 2008;
Huang et al., 2008). However, EO water with a neutral pH (NEW) can be produced. This can be done by redirecting part of the product formed at the anode side of the EO water generator (containing OH- ions) towards the cathode chamber during the electrolytic process (Pernezny et al., 2005; Guentzel et al., 2008). The resulting neutral solution has the following properties: 1) a pH ranging from 5.0 to 6.5; 2) hypochlorous acid is its most effective form of chlorine compound; 3) strong antimicrobial activity; 4) high ORP ranging from 700 to 800 mV; 5) less corrosive to processing equipment and irritation to the hands; 6) longer storage life than AEW because chlorine loss is greatly reduced; and
7) less hazardous to workers and environment when compared to NaOCl. This is so because it is produced by using salt, tap water, and electricity, thus, no concentrated chemicals are used in its manufacture (Cui et al. 2009; Hricova et al., 2008; Deza et al.,
2007; Ayebah and Hung, 2005; Len et al., 2002).
1.4.5.5.2. Ozone
Ozone is used mostly to treat drinking water and wastewater in Europe and in some US municipalities (Güzel-Seydim et al., 2004). This antimicrobial agent not only
39 has the generally recognized as safe (GRAS) status for bottled water treatment, it also has
GRAS status to be used in other food applications. For example, ozone has FDA approval to be used as a direct food additive for the treatment, storage, and processing of foods in gas and aqueous phases (Federal Register, 2001). Ozone, in gaseous form, is more frequently used for food storage/preservation applications. Some products currently being preserved with ozone include eggs during cold storage, fresh fruits and vegetables, and fresh fish (Perry and Yousef, 2011; Azarpazhooh and Limeback, 2008; Mahapatra et al.,
2005). In aqueous form, ozone is commonly used for the sanitization of surfaces, particularly of food processing equipment and packaging materials (Kim et al., 2003).
Ozone can be generated by several methods including ultraviolet (UV)-light radiation, corona discharge and electrochemical (cold plasma) techniques. During UV- light radiation, oxygen is exposed to UV light (wavelengths ranging from 140 to 190 mm), which splits the oxygen molecules into oxygen atom. The oxygen atoms then combine with other oxygen molecules to form ozone (Muthukumarappan et al., 2000).
This method results in relatively low concentrations of ozone (Kim et al., 1999).
Therefore, the corona discharged method is most commonly used, since it is capable of producing relatively high concentrations of ozone (Perry and Yousef, 2011; Mahapatra et al., 2005; Weddig et al., 2007).
A corona discharge ozone generator consists of two electrodes which are separated by a gas-filled discharge gap and a dielectric material usually made of glass or ceramic (Ölmez, 2012) (See Figure 1.3). In this method, gas (air or dry oxygen) is passed between these two high voltage electrodes, where high energy discharge splits molecular
40 oxygen into its atomic form. The atomic oxygen then spontaneously combines with molecular oxygen to form ozone (Perry and Yousef, 2011; Mahapatra et al., 2005).
Electrode
Voltage Dielectric surface ~ Oxygen Discharge gap Ozone
Dielectric surface
Electrode
Figure 1.3. Schematic diagram of corona discharge ozone generator (Kim et al., 1999).
The cold (non-thermal) plasma ozone generators are described as more efficient than the corona discharge generators (Akiyama et al., 2007). During ozone generation, pure oxygen is exposed to a plasma created by a dielectric discharge barrier (see Figure
1.4). A plasma can be defined as a gas containing charged and neutral species, including electrons, positive ions, negative ions, radicals, atoms, and molecules (Chan et al., 1996).
Therefore, the impact of energetic species in cold plasma treatments allows for the generation of ozone, since the mean energy of these species is higher than that of the ions and the neutrals in the oxygen gas (Akiyama et al., 2007). Unfortunately, cold plasma
41 generators are not frequently used for ozone generation due to the fact that they are much more expensive than the other types of generators (Ölmez, 2012).
High voltage electrode High voltage AC generator
Plasma discharge Dielectric ~ barrier
Ground electrode
Figure 1.4. Schematic representation of a cold plasma ozone generator (adapted from Niemira, 2012)
There are some advantages for the application of ozone in the food industry. For instance, when an excess of ozone is produced, it auto-decomposes to produce oxygen and leaves no residues in foods (Khadre et al., 2001). The ozone molecule is very unstable and decomposes, for instance, in aqueous solutions, into a number of oxidative
- radicals. These include hydroperoxyl (•HO2), hydroxyl (•OH), and superoxide (•O2 ) radicals. However, since the hydroxyl radical is highly reactive, the antimicrobial activity of ozone has been mainly attributed to the subsequent reaction of its decomposition products (Heim and Glas, 2011; Perry and Yousef, 2011). Therefore, ozone is a strong oxidizing agent (52% stronger than chlorine) that is effective over a much wider spectrum of microorganisms than chlorine and other sanitizers, including bacterial and
42 fungal spores and waterborne parasites (Van Houdt and Michiels, 2010; Weddig et al.,
2007). The antimicrobial activity of ozone appears to be similar to the chlorine-based sanitizers, which works by disruption of membrane permeability, impairment of enzyme function and/or protein integrity by oxidation of sulfhydryl groups and nucleic acid denaturation (Gerba, 2009; Stewart and Olson, 1996). However, several factors must be considered when ozone is used as a sanitizer. For example, the antimicrobial properties of ozone are affected by contact time, the presence of inorganic/organic matter, temperature and pH (with decreasing stability at increased temperatures and pH) (Castillo et al., 2003;
Kim, 1998). Also, ozone is highly reactive, readily corrodes metals, and degrades plastics and rubbers (Weddig et al., 2007). In addition to this, the investment costs of ozone generation systems are usually higher than other chemical sanitizing agents, although the running costs are considerably low as the only requirement is the electricity to produce the ozone (Pascual et al., 2007). Besides, human exposure to ozone can lead to a number of negative health effects if the ozone exposure is above certain levels. For example, low concentrations of ozone can irritate the respiratory system and cause headaches, coughing, dizziness, and nausea (Perry and Yousef, 2011).
1.5. Ware-washing protocols for the control of foodborne pathogens and the Food
Code
The FDA Food Code is an important document that provides practical, science- based guidance and enforceable provisions to retail outlets (e.g. restaurants and grocery stores) and institutions (e.g. nursing homes) on how to prevent foodborne illness. These
43 provisions are designed to be consistent with federal food laws and regulations, and are written for ease of legal adoption at all levels of government (Food Code, 2009). One of the provisions contained in the Food Code is the cleaning and sanitization of equipment and food contact surfaces (e.g. tableware). Ensuring effective cleaning and sanitization of these surfaces is an important function of food establishments. Contaminated surfaces
(e.g. utensils, cutting boards, equipment) have been identified as sources of cross- contamination for food during preparation and when being served to consumers. The spread of bacteria and viruses that can cause disease could be transmitted by these means.
According to the Food Code, a minimum microbial reduction of 5 logs must be obtained before surface sanitization could be considered effective. In order to achieve the standards set by the Food Code, restaurants and other food service establishments must clean and sanitize tableware items (e.g. dishes, glassware, and eating utensils) either manually or mechanically (McSwane et al., 2005). For the manual ware-washing operation, a three compartment sink is required for washing, rinsing and sanitizing of tableware items. The acceptable standards for manual ware-washing can be found in the
FDA Food Code (2009). For mechanical ware-washing operations, a mechanical
(automatic) dishwasher is employed. The American National Standard Institute (ANSI) and the National Sanitation Foundation (NSF) International provide the acceptable standards for mechanical ware-washing (ANSI/NSF 3, 2009). It is important to note that sinks and dishwashers used for the ware-washing of food contact surfaces should meet the standards and bear the stamp of approval from the (NSF) International. This will
44 assure that quality materials are used and that they are built according to acceptable standards (McSwane et al., 2005).
1.5.1. Manual versus mechanical ware-washing
These two ware-washing methods have similarities as well as differences. Both are design to render tableware free of soil and to achieve a minimum microbial reduction of 5 logs. It should be noted though that these methods specifically address the reduction of bacterial numbers from food contact surfaces, and not viruses. Therefore, information regarding their efficiency against viruses needs to be elucidated. Both ware-washing methods are designed to sanitize surfaces by using hot water or by applying a sanitizing solution (chemical sanitation). Some of the differences found within these two methods are: 1) the temperatures employed during the ware-washing procedure; and 2) the way they removed soils from surfaces. For instance, the temperature of the washing solution during manual ware-washing shall be at least 43ºC whereas for spray-type mechanical dishwashers that use sanitizing chemicals, it should be at least 49ºC. In the case of the sanitizing solutions, those commonly used during manual ware-washing are set at a minimum temperature of 24ºC. In contrast, chemical sanitization in mechanical dishwashers should be achieved at a minimum temperature of 49ºC. During manual ware- washing, the physical skill (mechanical action) of the employee is a key factor in the removal of soils (Tomlinson and Carnali, 2007). Generally, employees will use a brush or other approved implements to assist them in this task. In mechanical ware-washing, the mechanical action to remove soils is restricted by jets of water emitted from rotating
45 spray arms, the forces being much less than what results from the mechanical action of an individual during manual ware-washing. Therefore, in order to obtain good cleaning result, this water spray is compensated by the extra chemical action of the detergent, the temperature and time (Tomlinson and Carnali, 2007). Figure 1.5 summarizes the relative contributions of all factors during ware-washing procedures.
Mechanical action Chemical action 5% Temperature 15% 10% 25% Mainwash time 10%
25% 35% 75%
Machine dishwash contribution % Handwash Contribution %
Figure 1.5. Contribution of the key factors to cleaning for hand and machine dishwashing (Tomlinson and Carnali, 2007).
1.6. Adhesion of soils to surfaces and testing methods
Several factors are known to influence the adhesion of food soils to contact surfaces. These include physical and chemical characteristics of the food itself (e.g. viscosity, particle size), temperature, relative humidity and properties of the material used for the contact surface (Adhikari et al., 2001). The term adhesion has been previously
46 described as the physical phenomenon by which two materials, when in intimate contact, stick together (Michalski et al., 1997). Different theories have been used to explain the concept of adhesion, including thermodynamics (surface tension, contact angle), mechanics (rugosity, wear), and electrostatics (Michalski et al., 1998). However, for the purpose of this work, only the basics of the thermodynamic adhesion theory will be considered.
The strength of adhesion to a solid surface can be estimated from a value of the thermodynamic “work of adhesion” (Wa), defined as the reversible work per unit surface to separate two phases that initially have a common interface (Michalski et al., 1998).
This theory is based mainly on Young’s Force and Dupré Equations. Young described the relations between the contact angle and the forces acting on a liquid drop in mechanical equilibrium on a solid surface (Schrader, 1995). These forces relate solid and liquid surface tensions (γS) and work necessary to create one solid or liquid surface unity (γL), solid-liquid interfacial tension (γSL) work necessary to create one interface unity between solid and liquid, and the liquid contact angle (θ) at the solid/liquid/ air triple line
(Handojo et al., 2009). Dupre’s Equation, on the other hand, relates the work of adhesion
(Wa) to the molecular attraction between the liquid and that of the solid surface (Handojo et al., 2009; Adhikari et al., 2001). When a liquid drop is put on a solid/flat surface, it either forms a very thin film (complete wetting) or there is incomplete wetting of the solid by the liquid (von Rybinski, 2007). Good wettability means that a strong affinity exists between the liquid and the solid and these are likely to adhere well (Michalski et al., 1997). Contact angle is the inverse measure of wettability. Therefore, a lower contact
47 angle and higher wettability means a stronger attraction and the greater the forces required to separate these surfaces (Adhikari et al., 2001). Figure 1.6 provides a definition of the contact angle measurement when Young and Dupré Equations are combined and used to determine the work of adhesion (Wa) between a liquid and a flat solid surface with a known contact angle (θ).
Wa = γ (1 + cos θ) γL L
Vapor
θ Liquid γS
Solid γSL
Figure 1.6. Definition of contact angle (θ) from sessile drop geometry at solid/liquid/vapor triple point. γS, γL, and γSL are solid and liquid surface tension and solid-liquid interfacial tension, respectively (Michalski et al., 1998).
During ware-washing operations, the wettability concept is of great importance.
This is so because solid food soils are completely wetted only if cleaning solutions spontaneously spread on the contact surface (i.e. contact angle (θ) = 0) (von Rybinski,
2007). For most surfaces, the wetting tension is in agreement with the surface tension of the cleaners. Therefore, a spreading of the cleaning solutions on the surfaces and good wetting properties can be assumed (von Rybinski, 2007). One important aspect about
48 contact angle measurements is that, when used to estimate adhesions, it only provides an estimate of the difficulty involved in removing a given liquid product from an underlying surface. As a result, the measurements have to be taken before the product is removed
(Handojo et al., 2009). In addition to that, contact angle measurements can be complex to interpret, since they are valid only for an ideal solid (chemically homogeneous, rigid and flat at an atomic scale) (von Rybinski, 2007). Since most practical solid surfaces are rough to some extent, and may also be chemically heterogeneous and/or may contain surface active impurities in the liquid, these factors can lead to contact angle hysteresis
(von Rybinski, 2007; Chibowski et al., 2002). The hysteresis phenomenon is defined as the difference between the advancing contact angle (θa) (measured after the liquid front of the settled drop has progressed) and the receding contact angle (θr) (when the front has receded) (Chibowski et al., 2002). When contact angle hysteresis occurs, the angle measured on a liquid drop growing on the surface (advancing angle) is larger than the angle of a retracting drop (receding angle) (Michalski et al., 1997). Consequently, a measure of ambiguity in the determination of contact angle is then introduced under such circumstances (Della Volve et al., 2000).
1.6.1. Atomic Force Microscopy (AFM) for the analysis of surfaces
One factor to consider when the contact angle technique is used to estimate the adhesion strength of one material to a substrate is that the contact angle measurements have to be taken before the liquid soil under study is removed (Handojo et al., 2009).
Therefore, it is not practical to use this method when ware-washing procedures are
49 evaluated for their efficacy on the removal of food soils and microorganisms (e.g. bacteria and viruses). This is so, because the only way to accurately evaluate the performance of a ware-washing protocol is after it is completed. Atomic force microscopy (AFM) is a technique that can be used to study the adhesion strength between food soils and contact surfaces after ware-washing procedures. It is well known for its high resolution images of the surface topography of organic/inorganic films and biological materials (Herrmann et al., 2004; Raghavan et al., 2001). Also, AFM is capable of performing adhesion measurements under different environmental conditions and without special sample preparation (Méndez-Vila et al., 2002). It consists of a cantilever mounted tip, a piezoelectric scanner, four position-sensitive photodiodes
(photo-detector), a laser diode and a feedback control (Gaboriaud and Dufrêne, 2007).
The basic principle of AFM is to use the sharp tip scanning over the surface of a sample while sensing the interaction between that tip and the sample surface (Dufrene, 2008).
The interactions between the tip and the sample surface (commonly van der Waals forces) cause the cantilever to deflect (Liu and Cheng, 2010). van Der Waal forces occur only when atoms are very close together and they involve both attraction and repulsion forces (Horton et al., 2006). The attraction occurs when a pair of atoms approaches each other within a certain distance whereas the repulsion occurs when the distance between the interacting atoms becomes less than the sum of their contact radii (Li and Chou,
2003). When the AFM tip gets very close to the sample surface, the electron orbitals of the atoms on the surface of the tip and the sample start to repel each other, and as the gap
50 decreases, the repulsive forces neutralize the attractive forces (Beech, 1996). Thus, the position of the laser on the photo-detector changes (Gaboriaud and Dufrêne, 2007).
In order to measure the cantilever deflection with high resolution, the laser beam from the laser diode is usually focused on the free end of the cantilever, and the position of the reflected beam is thus detected by the photo-detector (Liu and Cheng, 2010). Once the laser is focused, the surface topography can be reconstructed, since the vertical position of the cantilever is established by sensing the short-range, repulsive portion of the interaction potential with the surface (McIntire et al., 1995). As the tip moves in response to the sample topography during scanning, the angle of the reflected laser beam changes and the laser spot falling onto the photo-detector moves, and this produces changes in intensity in each of its quadrants. Subsequently, the surface image of the sample is generated by a computer (Morris et al., 2010). Figure 1.7 illustrates the main features of the AFM technique.
51
Figure 1.7. Schematic representation of the atomic force microscope (Morris et al., 2010).
Different modes of operation can be used for AFM analysis including contact, tapping and non-contact. During contact mode, the tip and the atoms of the surface of the sample are in direct contact (the tip-sample interaction is repulsive in nature) (Morris et al., 2010; McIntire et al., 1995). The repulsion force between the tip and the sample surface causes the cantilever to deflect (Morris et al., 2010). Because the tip is in contact with the surface while scanning, a considerable shear force can be generated, causing damaging to the tip and the sample, especially in the case of very soft samples, due to capillary forces when imaging in air (Power et al., 1998). The contact mode can also be used to remove parts of a film or material by scratching the surface with a fixed relatively high force (Bhushan and Koinkar, 1994). This scratching method can be used to estimate 52 the thickness of a thin film attached to an underlying surface (Ton-That et al., 2000), particularly food soil films left on surfaces after ware-washing procedures. Figure 1.8 provides an illustration of how thin films or soft materials can be removed during contact mode.
Figure 1.8. Schematic of the scratch method with AFM tip on the surface of a sample (Choi et al., 2010)
During the tapping mode, the cantilever oscillates near its resonant frequency during the scan. Since the cantilever is deliberately excited by an electrical oscillator to amplitudes of up to ~ 100 nm, it bounces up and down (or taps) as it travels over the sample (Morris et al., 2010). The proximity of the scanned sample causes changes in the amplitude of oscillations of the probes due to van der Waals and electrostatic forces
(Gaczynska and Osmulski, 2008). Changes in the amplitude are translated into a
53 topography image offering a three-dimensional map of the surface of the object
(Gaczynska and Osmulski, 2008). One advantage of the tapping mode is that the image provides a better resolution of the topography of soft surfaces without damaging the surface (Handojo et al., 2009). For this reason, this mode can be employed to obtain an image of surfaces previously scratched (using the contact mode), thus allowing for film thickness measurements (Sigua et al., 2010; Handojo et al., 2009; Ton-That et al., 2000).
In non-contact mode the cantilever oscillates at a constant frequency above the sample, so the tip actually never touches the sample. However, due to van deer Waals forces and other long-range interactions extending above the surface, the motion of the tip is influenced and it generates information on the topography of the sample (Liu and
Cheng, 2010). Since there is no contact between the tip and the sample in this mode, the chances of damaging or contaminating the surface of the sample by the tip are low
(Morris et al., 2010). Therefore, the non-contact method can facilitate the analysis of biological materials where contamination and damaging of samples with the AFM tip is to be avoided (Kelsall et al., 2005).
1.6.2 Studies where AFM has been used to study the removal of food residues from food contact surfaces
Recently, the use of AFM in the field of food science has gained popularity, particularly in the area of food safety. The fact that AFM allows for film thickness measurements makes this technique a valuable tool to better study the efficacy of common ware-washing procedures for the removal of food soils from food contact
54 surfaces. Dairy-based products have been described as one of the most difficult types of soils to remove during ware-washing procedures (Lee et al., 2007). Therefore, surfaces soiled with dairy-based products may require extra effort to be effectively cleaned and sanitized. Handojo et al. (2009) used the AFM technique to estimate the adhesion strength of milk-based samples on glass surfaces and to determine the thicknesses of the films from each sample after manual ware-washing protocol. Likewise, Sigua et al.
(2010) used AFM to measure the efficacies of various chemical sanitizers in removing milk-based samples from glass surfaces and to quantify the thicknesses of the milk films left on surfaces after mechanical ware-washing. These studies confirmed that the AFM technique can be used to estimate the difficulty of removing milk-based products from food contact surfaces. However, information regarding food contaminated with pathogens and the influence these pathogens may have on the removal of food soils after ware-washing procedures is limited. Therefore, AFM can assists to study the role different microorganisms (e.g. bacteria and viruses) may have on the adhesion of foods to surfaces in order to generate valuable information that might lead to the evaluation or improvement of common ware-washing protocols. Besides, information obtained from
AFM studies could be used to determine the efficacy of different detergent and sanitizing solutions on soiled food contact surfaces during ware-washing procedures, which could help to formulate and test a broader range of products.
55
1.7. References
Abbas, S, Goldberg, JW and Massaro, M. 2004. Personal cleanser technology and clinical performance. Dermatologic Therapy, 17:35-42.
Adhikari, B, Howes, T, Bhandari, BR and Truong, V. 2001. Stickiness in foods: a review of mechanisms and test methods. International Journal of Food Properties, 4:1-33.
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CHAPTER 2: EFFICACY OF SODIUM HYPOCHLORITE AND QUATERNARY
AMMONIUM COMPOUNDS DURING WARE-WASHING OPERATIONS FOR
SANITIZATION OF TABLEWARE ITEMS CONTAMINATED WITH
NOROVIRUS AND SELECTED BACTERIA
2.1. Abstract
Cross-contamination of ready-to-eat (RTE) foods with pathogens on contaminated utensils is an important factor associated with foodborne illnesses. This study evaluated the sanitization efficacies of ware-washing protocols (manual and mechanical) used in restaurants to clean tableware items contaminated with murine norovirus (MNV-1).
Escherichia coli K-12 and Listeria innocua were also investigated and their survivability and response to the experimental conditions were compared with that of MNV-1. The sanitizing solutions tested were sodium hypochlorite (chlorine), quaternary ammonium
(QAC) and tap water (control). Ceramic plates, drinking glasses and stainless steel forks were used as the food contact surfaces. Results showed that current ware-washing protocols used to remove bacteria from tableware items were not enough to produce a 5 log reduction in MNV-1. After the ware-washing protocols, a maximum of 3 log reduction in the virus was obtained. It was concluded that MNV-1 appeared to be more
82 resistant to both washing protocols and the sanitizers when compared with E. coli K-12 and L. innocua.
2.2. Introduction
Norovirus (NoV) is the leading cause of epidemic gastroenteritis and the major cause of foodborne illness in the United States. It is responsible for at least 50% of all gastroenteritis outbreaks worldwide (Hall et al., 2011). NoV is highly contagious and only a few particles are sufficient to cause illness (Teunis et al., 2008; Estes et al., 2006;
Duizer et al., 2004). Transmission of the vast majority of foodborne NoV infections is considered to be through the oral-fecal route, either by direct person-to-person spread or indirectly through contaminated food or water (Moe, 2009; Goodgame, 2007; Koopmans and Duizer, 2004). Common foods associated with the transmission of NoV include fresh produce, ready-to-eat foods, oysters, baked goods, and berries (Gibson and Schwab,
2011). Ingestion of aerosolized vomitus, indirect exposure via fomites or contaminated environmental surfaces are also recognized as important means for the transmission of
NoVs (Hall et al., 2011).
Restaurants and foodservice establishments are recognized as important sites for the transmission of foodborne illnesses (Verhoef et al., 2009; Goodgame, 2007; Hedberg et al., 2006). This is so because of the large numbers of individuals who patronize these places (NRA, 2010). As part of a 10 year study which began in 1998, the Food and Drug
Administration (FDA) collected data from more than 800 food establishments. This study evaluated the occurrence of practices and behaviors commonly identified by the Centers
83 for Disease Control and Prevention (CDC) as contributing factors to foodborne illness outbreaks (FDA, 2009a). At the conclusion of the study, five foodborne illness risk factors were identified. These risk factors include: 1) food from unsafe sources; 2) poor personal hygiene; 3) inadequate cooking; 4) improper holding temperatures; and 5) contaminated equipment. The report also showed that contaminated equipment had a high percentage of out of compliance observations. Under this category, improper cleaning and sanitizing of food-contact surfaces was the item most commonly observed to be out of compliance.
A previous study conducted by Handojo et al., (2009) showed that traditional sanitizers were able to reduce Escherichia coli K-12 and Staphylococcus epidermidis by
≥ 5 log, the minimum reduction required by the FDA Food Code for an effective sanitization protocol for bacteria (FDA, 2009b). However, little information regarding the efficacy of traditional sanitizers for the reduction of foodborne viruses from food contact surfaces is available in the literature. The objective of this study was to evaluate the sanitization efficacy of quaternary ammonium and sodium hypochlorite for the reduction of murine norovirus (human NoV surrogate) on different contaminated tableware items using normal ware-washing protocols (manual and mechanical), and compare it with the response for bacteria species (E. coli K-12 and L. innocua) under similar conditions.
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2.3. Materials and Methods
2.3.1 Cell culture and virus stock
The MNV-1 was provided by Dr. Herbert Virgin IV from Washington University
School of Medicine. The preparation and infectious titer assays for MNV-1 were performed using mouse monocyte macrophage cell line RAW 264.7 (ATCC, Manassas,
VA), as described by Wobus et al., (2006), with minor modifications. The cells were cultured and maintained in 150 cm2 tissue culture flasks (BD Falcon, Bedford, MA) containing high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen,
Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; GIBCO-Invitrogen,
Grand Island, NY) at 37ºC and 5% CO2 atmosphere. Confluent RAW 264.7 cells were infected with the MNV-1 at a multiplicity of infection (MOI) of 0.2. The flasks were incubated at 37ºC and 5% CO2 for 1 h, with agitation every 15 min. After 1 h incubation,
DMEM supplemented with 2% FBS was added to the flask and incubated at 37ºC and 5%
CO2 for 48 h. When extensive cytopathic effect (CPE) was observed, the virus was harvested by freeze-thawing three times at these temperatures -80ºC and 37ºC, respectively, to lyse the cells and release virus particles. The purification of MNV-1 was performed by transferring the cell-virus suspensions into 50 ml sterile conical centrifuge tubes (USA Scientific, Ocala, FL) and centrifugation at 3,000 rpm for 20 min using an
Allegra 6R centrifuge with a GH-3.8 swinging bucket rotor (Beckman Coulter, Brea,
CA). The supernatant was collected and stored at 4ºC for immediate use or at -80ºC in aliquots for future use.
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2.3.2. Preparation of bacterial cultures
E.coli K12 (ATCC 29181) and L. innocua (ATCC 33090) were stored in a - 80ºC freezer in 30% (v/v) sterile glycerol (Fisher Scientific, Fair Lawn, NJ) and revived when required for the experimental procedure. The stock culture of each organism was prepared by transferring a loopful of the E. coli and L. innocua into 50 ml of Trypticase soy broth (Difco, Becton Dickinson, Sparks, MD) containing 0.3% (wt/wt) yeast extract
(Fisher Scientific) (TSBYE) and then incubation of the cultures at 37ºC for 24 h. A loopful of this broth was then inoculated into a Trypticase soy agar slant (Difco, Becton
Dickinson) supplemented with 0.3% (wt/wt) yeast extract (TSAYE) and incubated at
37ºC for 24 h. This TSAYE containing the cell cultures was stored in a refrigerator at 3ºC and used as a stock culture.
Prior to each experiment, a loopful of either E. coli K12 or L. innocua stock culture was propagated aerobically in 100 ml TSBYE at 37ºC for 24 h. Each cell broth was centrifuged (Kendro Laboratory Products, Sorvall RC 5C Plus, Newtown, CT) at
6,000 rpm for 10 min at 4ºC. The supernatant was decanted and the cell suspension was resuspended in 100 ml 0.1 M 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 was achieved. Each cell suspension was separately mixed with the food samples to be tested.
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2.3.3. Food samples preparation and inoculation
Cream cheese spread and 2% reduced fat UHT milk were used to contaminate the food contact surfaces in this study. These were purchased from a local grocery store in
Columbus, OH. They were both stored in a refrigerator at 4ºC until ready to use. Ceramic plates, stainless steel forks and drinking glasses were the tableware items tested. These were sterilized by autoclaving at 121ºC for 20 min before each experiment.
2.3.3.1. Ceramic plates
For the contamination of the ceramic plates, 90 g of cream cheese were weighed in a sterile beaker, heated for less than 10 sec in a microwave, inoculated with 10 ml of the virus stock (1:10 w/w) to provide a final titer of approximately 7 log plaques forming units per gram (PFU/g). This was then stirred with a sterile tongue depressor (Fisher
Scientific, Florence, KY) to ensure proper mixing of the virus with the cream cheese. A total of 3 g of this cream cheese was then applied to the entire food contact surface of each ceramic plate. The plates were air dried for 1 h at room temperature (25ºC) on a flat, sterile rack prior to the washing protocol. The same procedure was followed for E. coli
K- 12 and L. innocua.
2.3.3.2. Forks and drinking glasses
From the contaminated cream cheese above, a 0.5 g aliquot was applied to the fork. For contamination of the glasses, 45 ml of milk were transferred to a 50 ml sterile conical tube and inoculated with 5 ml of virus stock solution (1:10 v/v). From this
87 solution, 0.5 ml was applied to the inner wall of each drinking glass. Both the forks and the glasses were then air dried for 1 h at room temperature (25ºC) on a flat, sterile rack prior to the washing protocol. The same procedure was followed for E. coli K- 12 and L. innocua.
2.3.4. Preparation of the detergents and sanitizing solutions
Two different detergents were used during the washing step. Ecotemp Ultra Klene detergent (Ecolab, Inc., St. Paul, MN) was used for the mechanical washing whereas
Monsoon detergent (Ecolab, Inc., St. Paul, MN) was used for the manual washing. Ultra
Klene detergent was used at 3,000 ppm concentration and its ingredients included sodium hydroxide, sodium polyacrylate and sodium phosphonobutanetricarboxylate. The concentration used for the Monsoon detergent was 100 ppm and it contained linear alcohol (C12-C15), decylamine oxide, linear alcohol (C9-C11) and lauryl dimethylamine oxide as its main ingredients. These concentrations were recommended by the manufacturer, as per the Food Code requirements.
Two different sanitizers were used during this study. These were sodium hypochlorite (chlorine-bleach) and quaternary ammonium compounds (QAC). The chlorine bleach, containing 6% sodium hypochlorite, was purchased from a local grocery store. The sodium hypochlorite solution used in this study was 200 ± 20 ppm and this concentration was determined using a HI 95771 Chlorine Ultra High Range Meter
(Hanna Instruments, Ann Arbor, MI).
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The QAC was an OASIS 146 Multi-Quat sanitizer manufactured by Ecolab, Inc.
(St. Paul, MN). The ingredients in this sanitizer were n-Alkyl (50% C14, 40% C12, 10%
C16), dimethyl benzyl ammonium chloride (3.000%), octyl decyl dimethyl ammonium chloride (2.250%), didecyl dimethyl ammonium chloride (1.125%) and dioctyl dimethyl ammonium chloride (1.125%). It was used at a concentration of 200 ppm as determined by a HYDRION QT-10 Quat test paper (QA Supplies, Norfolk, VA). According to the
FDA Code (2009), QAC sanitizers must be mixed with water with a hardness level less than 500 ppm CaCO3. To ensure compliance with this, the water hardness was determined using a Water Quality Test Strip kit (Hach Co., Loveland, CO). The water hardness for both mechanical and manual washing procedures was determined to be less than 120 ppm.
2.3.5. Mechanical ware-washing and sanitization of contaminated tableware items
For the mechanical ware-washing study, an LXiC Dishwasher manufactured by
Hobart Corp. (Troy, OH) was used. This was connected to a hot water line and had an incoming water pressure of 138 kPa. The hardness of this water was determined to be 120 ppm by a Water Quality Test Strip kit (Hach Co., Loveland, CO). Prior to each experiment, the machine was cleaned with hot water 49°C and filled with fresh water.
The dishwasher water tank held approximately 11.4 liters. To ensure the proper volume and concentration of the detergent during the washing cycles, the detergent (Ecotemp
Ultra Klene detergent) was directly added to the water tank. The resulting water- detergent solution had a concentration of 3,000 ppm (v/v). During the washing cycle, the
89 tableware items were automatically sprayed with the wash water for 76.5 s at a pressure and temperature of 138 kPa and 49°C, respectively. After the washing cycle, the tableware items were automatically sprayed with the QAC sanitizing solutions for 10 s at
49°C. Once the sanitizing cycle was completed, all tableware items were air dried for 1 h at 24 ± 2°C. This entire cycle was repeated but with the sodium hypochlorite sanitizer instead.
2.3.6. Manual ware-washing and sanitization of contaminated tableware items
For the manual dishwasher, a three compartment sink, manufactured by Eagle
Group, Inc. (Clayton, DE), was used for the washing, rinsing and sanitizing of the tableware items (Figure 2.1). Prior to each experimental run, this dishwasher was thoroughly cleaned with hot water and refilled with fresh water and detergent/sanitizer.
Each of the three compartments was filled with 28 liters of water. The tableware items were washed with 100 ppm of the Monsoon detergent at 43°C for 30 s, rinsed with tap water at 24°C for 10 s, and sanitized with one of the sanitizing treatments at 24°C for 30 s. The test was repeated for each sanitizer and tableware item. Rubber gloves were worn throughout the experiment.
During the washing step, each ceramic plate and fork was manually washed using a Scotch-Brite multi-purpose scrub sponge (3M, St. Paul, MN). To ensure consistency of the force applied to remove the cream cheese from the plates and the forks during washing, the sponge was attached to a spring-loaded device (Figure 2.2 [b]). A cylindrical device covered with a soft sponge was used to wash the drinking glasses
90
(Figure 2.2 [a]). The forks were washed by using fifteen forward and fifteen backward strokes with the sponge. The plates and glasses were washed by using fifteen clockwise and fifteen counter-clockwise strokes. After washing, the tableware items were rinsed, sanitized and placed in a clean rack and air dried for 1 h at 24 ± 2°C. The three compartments of the sink were washed with hot water (49ºC) and bleach (10%) after each experimental run.
Figure 2.1. Experimental design of the mechanical and manual ware-washing protocols followed for the removal of MNV-1, E. coli K-12 or L. innocua from different tableware items.
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Figure 2.2. Devices used during manual ware-washing to clean the different tableware items. a) Cylindrical sponge b) Sponge attached to the spring-loaded tool.
2.3.7. Viral sampling of the tableware surfaces: MNV-1 plaque assay
The initial viral titer of the virus stock, as well as from the contaminated milk, was determined by transferring 200 μl from each to a test tube containing 1.8 ml phosphate buffered saline (PBS). The titer of the contaminated cream cheese was determined by transferring a small amount of the cheese to a test tube containing 2 ml of
PBS using a sterile calcium-alginate cotton-tipped swab (Fisher Scientific, Pittsburgh,
PA). Prior to the ware-washing, 4 samples of each tableware item were collected after the air drying period. Once the sanitization step was completed and the tableware items air dried for 1 h, 4 samples of each item were collected. These samples (before and after the ware-washing procedure) were collected using cotton-tipped swabs moistened with the
PBS solution. The test tubes containing the samples were vortexed to remove any viral particles attached to the tip of the swab. Serial dilutions (10-fold) of the samples were performed in PBS solution.
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The quantification of the MNV-1 was performed by plaque assay. Confluent monolayers of RAW 264.7 cells were grown in 6-well plates (BD Falcon, Franklin
Lakes, NJ) containing DMEM with 10% FBS for 24 h at 37ºC and 5% CO2. After incubation, the growth medium was removed and the cell monolayers were infected with
500 μl of each sample dilution. The infected plates were incubated for 1 h at 37ºC and 5
% CO2, with agitation every 15 min. The plates were overlaid with 2 ml minimal essential medium (MEM) supplemented with 5% FBS, 1.6% sodium bicarbonate (7.5%
[wt/v]), 0.5% penicillin-streptomycin (10,000 U of penicillin and 10,000 μg/ml streptomycin in 0.85% saline; GIBCO-Invitrogen, 2.5% HEPES, 1% glutamine, and
1.5% low-melting-point agarose (GIBCO-Invitrogen). After adding the overlay, the plates were placed in a refrigerator (4ºC) for 1 h and incubated at 37ºC and 5% CO2 for 2 d. Following this, 2 ml of 10% formaldehyde in PBS solution were added to each well to fix the cells. The fixation was done for 4 h. The overlay-formaldehyde solution was removed and the wells stained with 0.05% crystal violet (wt/v) for 1 h in order to visualize and quantify the viral plaques.
2.3.8. Bacterial enumeration of the contaminated tableware surfaces
For enumeration of the bacteria, the samples before and after the ware-washing procedure were collected using the cotton-tipped swabs, previously moistened in 0.1% peptone water solution. The swabs were then transferred to test tubes containing 0.1% peptone water and vortexed vigorously to remove any bacteria from the tips.
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The total viable counts were determined by serial dilution of the samples using test tubes and then plated into TSAYE. The plates were incubated at 37ºC for 36 h. A
Darkfield colony counter (American Optical, Buffalo, NY) was used to count the bacterial cells. The detection limit for estimating the bacterial numbers was 2 CFU per tableware item.
2.3.9. Statistical analysis
All tests were duplicated in this study. The viral counts were expressed as log
PFU per tableware (surface). For the bacterial cells, the counts were expressed as log
CFU per tableware. During each test, 4 tableware items were selected for viral/bacterial enumeration before and after sanitization. The reported values of the viral counts were the mean values of two trials ± standard deviations. Multifactor analysis of variance
(ANOVA) was used to determine the significance between the mean values. The data analyses were performed by the General Linear Model function and Tukey’s multiple comparison test with the SAS, version 9.2, statistical program (SAS Institute, Cary, NC) to determine the level of significance between the effect of each sanitizer, tableware item and ware-washing protocol (manual vs. mechanical). To properly identify any significant differences at low levels, the p value was set at < 0.0001.
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2.4. Results and Discussion
2.4.1. Effect of air-drying on the reduction of MNV-1, E. coli and L. innocua on the contaminated tableware
Prior to each ware-washing experiment, all contaminated tableware items were allowed to air-dry for 1 h. This simulated the average time tableware items may sit in a restaurant before washing. It also provided sufficient time for the contaminated food to adhere to the tableware items. Figure 2.3 shows the effect of this air-drying on the initial
MNV-1 counts. The highest mean reduction in the viral counts was 0.1 log PFU per tableware item. The statistical analysis confirmed that the viral counts on the contaminated tableware items before and after the air-drying period were not significantly different (p > 0.0001). This result is consistent with those of other researchers who showed that NoV can survive for up to 30 days on stainless steel
(Takahashi et al., 2011) and 7 days on fecal contaminated surfaces (Cannon et al., 2006 and D’Souza et al., 2006). This suggests that MNV-1 is quite resistant to air-drying and that it could remain viable on food contact surfaces for an extended period of time.
Figures 2.3 also shows the effect of the air-drying on the initial counts of E. coli
K-12 and L. innocua cells. The highest reductions observed for E. coli K-12 (0.9 log) and
L. innocua (0.4 log) were on the plates The mean reductions on the forks and drinking glasses for both organisms ranged between 0 to 0.4 log. Overall, these results are in agreement with previous studies (Handojo et al., 2009; Lee et al., 2007), where E. coli K-
12 and L. innocua showed stability under drying conditions. These results also show that
E. coli and L. innocua are more sensitive to desiccation stresses than MNV-1.
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Plates Forks Glasses
10.0
9.0
8.0
7.0
6.0
5.0
4.0 PFU or CFU/ tablewareor PFUCFU/
10 3.0
Log 2.0
1.0
0.0 Pre-Drying Post- Drying Pre-Drying Post- Drying Pre-Drying Post- Drying MNV-1 E.E. coli coli L.L. innocua innocua
Figure 2.3. Survival of MNV-1, E. coli K-12 and L. innocua on different contaminated tableware before and after 1 hour air-drying at 24 ± 2°C.
2.4.2. Comparison between the efficacies of the mechanical and manual ware- washing protocols and the effect of the sanitizers
In order to meet the Food Code (2009b) specifications for proper washing and sanitization of tableware in a mechanical dishwasher using chemical sanitizers, the temperatures during washing and sanitization must be at least 49ºC.
Figure 2.4 shows the survivability of MNV-1 on the contaminated surfaces before and after mechanical washing. Results showed that the mean reductions of MNV-1 on the plates, forks and drinking glasses after washing treatment with the control were 2.6, 1.3 and 0.7 log, respectively. The mean reductions achieved after washing and chlorine
96 sanitation (3.2, 1.5 and 1.4, respectively) were slightly higher than those obtained by the control treatment. Statistically, chlorine reductions were not significantly different (p >
0.0001) from those achieved by the control. Similarly, the mean reductions achieved by the QAC sanitizer were not significantly different (p > 0.0001) from the reductions produced by the control and chlorine treatments. The data showed that after washing and sanitizing with the QAC sanitizer MNV-1 was reduced on the ceramic plates by 2.7 log and the mean reduction for both the forks and glasses were 1.6 and 1.4 log, respectively.
Overall, the viral counts detected on the different surfaces after sanitization with the three treatments were statistically different (p < 0.0001) than the initial viral counts prior to the ware-washing. When comparing the mean reductions achieved for MNV-1 on the three different surfaces, the data show that they were not statistically different (p > 0.0001).
97
Plates Forks Glasses 9.0
8.0
7.0
6.0
5.0
4.0
3.0 PFU or CFU/ tableware or PFUCFU/
10 2.0 Log 1.0
0.0
QAC QAC QAC
Wash Wash Wash
- - -
Control Control Control
Chlorine Chlorine Chlorine
Pre Pre Pre MNV-1- Mechanical E.E. coli coli K K-12-12- MechanicalMechanical L.L. innocua innocua- -MechanicalMechanical
Figure 2.4. Survival of MNV-1, E. coli K-12 and L. innocua on contaminated tableware items after washing and sanitizing, using the mechanical dishwasher.
During the manual ware-washing, the minimum temperatures were 43ºC, 24ºC and 24ºC for the washing, rinsing and sanitizing solutions, respectively. These temperatures conform to the requirements set by the Food Code (2009b). To minimize variability in this study, only one individual manually washed the tableware items.
The effect of the manual ware-washing and sanitizing solutions on the reduction of MNV-1 from the contaminated tableware items is presented in Figure 2.5. For the control treatment, the mean reductions of MNV-1 on the plates, forks and glasses were
2.8, 1.1 and 1 log, respectively. The reductions achieved by the chlorine and the QAC sanitizers were slightly higher than the ones obtained by the control. For chlorine sanitization, the reductions ranged from 1.7 to 3.5 log per tableware item. For the QAC 98 sanitizer, the range was 1.6 to 3.2 log per tableware item. The statistical analysis revealed that the mean reductions achieved after sanitization with the chlorine and QAC sanitizers were not significantly different (p > 0.0001) than the reductions achieved by the control.
In general, the viral reductions achieved after manual ware-washing appeared to be a little larger than those achieved after mechanical ware-washing.
Plates Forks Glasses 9.0
8.0
7.0
6.0
5.0
4.0
3.0 PFU or CFU/ tableware or PFUCFU/
10 2.0
Log 1.0
0.0
QAC QAC QAC
Wash Wash Wash
- - -
Control Control Control
Chlorine Chlorine Chlorine
Pre Pre Pre MNV-1- Manual E.E. coli coli K K-12-12- -ManualManual L. L.innocua innocua- Manual- Manual
Figure 2.5. Survival of MNV-1, E. coli K-12 and L. innocua on contaminated tableware items after washing and sanitizing, during manual ware-washing.
The effect of the ware-washing and sanitizing solutions on the reduction of E. coli
K-12 and L. innocua from the contaminated tableware items are also presented in Figures
2.4 and 2.5. In general, bacterial cells were significantly (p < 0.0001) reduced after the 99 tableware items were washed and sanitized. However, the data show that the mechanical ware-washing produced slightly more inactivation of viable cells when compared with the manual method (Figures 2.4 and 2.5, respectively). This could be attributed to the water pressure in the automatic dishwasher as well as the higher temperature used during the washing cycle. This may have synergistically helped to break down the adhesion bonds between the food residues and the surfaces (Handojo et al., 2009). All sanitizing solutions during the mechanical ware-washing were able to produce ≥ 5 log reduction.
Additionally, the results suggest that when the chlorine solution is used during mechanical ware-washing, the reduction of E. coli K-12 from the plates tends to be higher when compared with that of the forks and the drinking glasses.
Most documented foodborne viral outbreaks can be traced to food that has been manually handled by an infected food handler (Koopmans and Duizer, 2004). Hence, the hygiene of the personnel who handle food in foodservice establishments is an important preventive measure in minimizing cross-contamination of food contact surfaces and the food itself with NoV (D’Souza et al., 2005; Bean et al, 1996). Additionally, the long persistence of NoV on food preparation surfaces and its resistance to heat and disinfection, make the issue of cross-contamination reduction an even more urgent matter in the fight against foodborne outbreaks (D’Souza et al., 2006; Kusumaningrum et al.
2003).
Results presented in Figures 2.3 and 2.4 reveal that even though sanitizers appear to slightly enhance the reduction of MNV-1 from contaminated tableware, there was still a considerable amount of the virus on the contaminated surfaces. Since NoV is highly
100 contagious and its infectious dose is relatively low (10-100 particles), only a few infectious virus particles can cause human infection (Hall et al., 2011; Teunis et al., 2008;
Cheesbrough et al., 2000). In accordance with the guidelines provided by the ANSI/NSF
International standards (ANSI/NSF 3) and the FDA Food Code (2009b), any chemical sanitizing treatment used during ware-washing operation should achieve a 5-log microbial reduction. Unfortunately, these mandates are based on studies designed for the reduction of bacterial populations, but not viruses. Therefore, based on the results obtained in this present study, viruses such as MNV-1 seem to be quite resistant to the common sanitizers used in restaurants and other foodservice facilities. In addition to this, ware-washing protocols that previously showed effectiveness in removing a significant amount of bacteria from contaminated food contact surfaces (Handojo et al., 2009), appear not to have the same efficacy for the removal of viruses.
There are some possible explanations regarding the ineffectiveness of the ware- washing procedures to achieve higher reductions of the virus from the contaminated surfaces. One could be the food itself. Food residues are known to protect bacteria from direct contact with the heat or detergents used in dishwashing operations
(Kusumaningrum et al., 2002; Line et al., 1991). Likewise, inactivation studies suggests that food matrices may also provide a protective effect for virus inactivation (Lou et al.,
2011; Sanglay et al., 2011; D’Souza et al, 2006). The food matrices used in our study were cream cheese and 2% reduced fat milk. Generally, milk and milk products are more difficult to remove from eating utensils than other types of foods (Lee et al., 2007).
Cream cheese has a high fat content and most of its ingredients are not soluble in water
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Consequently, when fats are adhered to a food contact surface, it will be more difficult to remove these residual foods if an appropriate solvent is not employed (Sigua et al., 2011).
Although milk, on the other hand, is more soluble in water, it also contains some fat and proteins, which might act as protective agents as well (Hirneisen et al., 2010; Chen et al.,
2004). Another explanation for the persistence of MNV-1 on the surfaces could be the drying time prior to the experiment. All contaminated surfaces were allowed to dry for 1 hr and it is possible that while slowly drying, they have formed a layer that protected the virus on the surfaces, resulting in a prolonged survival (Kusumaningrum et al., 2003).
The formation of this layer can also be the reason for the detection of E. coli and L. innocua in our present study, even after ware-washing. With this in mind, it can be assumed that food residues, not visibly detected, could harbor infectious virus/bacteria and protect them from the effect of the sanitizers.
The ineffectiveness of the traditional sanitizers and the ware-washing protocols to remove and/or inactivate MNV-1 from contaminated surfaces is in agreement with published reports which suggested that the current food hygiene guidelines, most of which have been optimized for the prevention of bacterial infections, may be only partially effective against viruses (FAO/WHO, 2008; Koopsmans and Duizer, 2004).
Additionally, previous viral inactivation studies have also shown that non-enveloped viruses (e.g. NoVs and their surrogates) are fairly resistant to chemical agents, including
QACs and chlorine-based sanitizers (Nowak et al., 2011; Malik and Goyal, 2006; Gulati et al., 2001). The poor virucidal activity of QAC against MNV-1 in our study could be attributed to its formulation and the types of microorganisms it is intended to kill. Nowak
102 et al. (2011), Whitehead and McCue, (2010) and Eleraky et al. (2002), explained this by noting that when used alone, QAC sanitizers have limited effectiveness for inactivation of non-enveloped viruses such as NoV and its surrogates. However, modifications to
QAC by incorporation of additives such as sodium carbonate coupled with increments in its pH, enhances its virucidal activity (Whitehead and McCue, 2010; Jimenez and
Chiang, 2006; Gulati et al., 2001). Common quaternary-based sanitizers are formulated in such a way that they are able to kill bacteria and some enveloped viruses (Hegstad et al.,
2010). This selectivity of QAC has been attributed to its ionic binding capabilities and hydrophobic interactions with microbial membrane surfaces. This is so because QAC is positively charged and when in contact with microorganisms its cationic head is oriented outwards and the hydrophobic tail attracted to the lipid bilayers of the organisms. This causes rearrangement of the membranes and subsequently leakage of the intracellular constituents (Ioannou et al., 2007). However, the bactericidal activity of QAC can be reduced when bacteria are allowed to dry with foods on surfaces. This is supported by a previous study conducted by Kuda et al. (2008), where food sediments, particularly milk, adversely affect the bactericidal effect of QAC. Therefore, it is not surprising that the
QAC sanitizer in our current study did not completely eliminate E. coli and L. innocua from the tableware items, although it was able to achieve a 5 log reduction.
Even though the MNV-1 viral capsid is negatively charged, it is composed mainly of proteins. Because of this, it should be expected that chemical sanitizers such as hypochlorites should possess strong virucidal activities against non-enveloped viruses such as MNV-1. This is so because they react strongly with amino or sulfhydryl groups
103 that are present in the organisms (Gerba, 2009; Stewart and Olson, 1996). Despite this, the results from our study showed that during both mechanical and manual ware-washing protocols, chlorine showed little effectiveness in reducing the MNV-1 on the contaminated surfaces. However, chlorine was able to inactivate both E. coli K-12 and L. innocua. One possible explanation for the failure of the chlorine sanitizer in our study to cause a greater viral reduction is the pH of the sanitizing solution. The pH of our solution was approximately ≥ 7.5. However, hypochlorous acid (HOCl), which creates a more acidic pH tends to be more aggressive in killing bacteria (Cheng and Hsieh, 2010;
Christensen et al., 2008). Although sodium hypochlorite solutions are also expected to show some antimicrobial effect because of the presence of hydroxide ions (OH-) at pH over 11, these solutions are less effective than those of more acidic pH due to the presence of hypochlorite ions (OCl-), also known for their poor antimicrobial activity
(Estrela et al., 2002; Rutala and Weber, 1997; Springthorpe and Sattar, 1990). Thus, the pH of the chlorine sanitizing solution in our study may have played an important role in the ineffectiveness against infectious MNV-1, since the main active species of hypochlorite was in the form of OCl-. It is worth mention that although sodium hypochlorite solutions at more acidic pH might enhance the removal of non-enveloped viruses from food contact surfaces, these solutions are highly corrosive to equipments, which is the main reason they are mainly used at neutral pH (FDA, 2001).
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2.5 Conclusions
From the results of this study, it could be concluded that QAC and sodium hypochlorite sanitizers normally used to inactivate bacteria in manual and mechanical ware-washing operations are unable to produce the same level of antiviral activity under similar conditions irrespective of the nature of the tableware item.
105
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Bean, NH, Goulding, JS, Lao, C and Angulo, FJ. 1996. Surveillance for foodborne- disease outbreaks -United States. 1988-1992. Morbidity and Mortality Weekly Report (MMWR) -CDC Surveillance Summaries, 45:1-66.
Cannon, JL, Papafragkou, E, Park, GW, Osborne, J, Jaykus, LA, Vinjé, J. 2006. Surrogates for the study of norovirus stability and inactivation in the environment: A comparison of murine norovirus and feline calicivirus. Journal of Food Protection, 69:2761-2765.
Cheesbrough, JS, Green, J, Gallimore, CI, Wright, PA and Brown, DW. 2000. Widespread environmental contamination with Norwalk-like viruses (NLV) detected in a prolonged hotel outbreak of gastroenteritis. Epidemiology and Infection, 125:93-98.
Chen, H, Joerger, RD, Kingsley, DH and Hoover, DG. 2004. Pressure inactivation kinetics of phage λ cI 857. Journal of Food Protection, 67:505-511.
Cheng, H-H and Hsieh, C-C. 2010. Integration of chemical scrubber with sodium hypochlorite and surfactant for removal of hydrocarbons in cooking oil fume. Journal of Hazardous Materials, 182:39-44.
Christensen, CE, McNeal, SF and Eleazer, P. 2008. Effect of lowering the pH of sodium hypochlorite on dissolving tissue in vitro. Journal of Endodontics, 34:449-452.
D’Souza, DH, Sair, A, Williams, K, Papafragkou, E, Jean, J, Moore, C, and Jaykus, L. 2006. Persistence of caliciviruses on environmental surfaces and their transfer to food. International Journal of Food Microbiology 108:84-91.
Duizer, E, Bijkerk, P, Rockx, B, de Groot, A, Twisk, F and Koopmans, M. 2004. Inactivation of caliciviruses. Applied and Environmental Microbiology, 70: 4538-4543.
Eleraky, NZ, Potgieter, LND and Kennedy, MA. 2002. Virucidal efficacy of four new disinfectants. Journal of the American Animal Hospital Association, 38:231-234.
Estes, MK, Prasad, BV and Atmar, RL. 2006. Noroviruses everywhere: Has something changed? Current Opinion in Infectious Diseases, 19:467-474.
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Estrela, C, Estrela, CRA, Barbin, EL, Spanó, JCE, Marchesan, MA and Pécora, JD. 2002. Mechanism of action of sodium hypochlorite. Brazilian Dental Journal, 13:113-117.
Food and Agriculture Organization of the United Nations/ World Health Organization (FAO/WHO). 2008. Viruses in food: scientific advice to support risk management activities. Meeting Report, Microbiological Risk Assessment Series 13. Accessed on 4/19/12. Available at: http://www.who.int/foodsafety/publications/micro/Viruses_in_food_MRA.pdf
Food and Drug Administration (FDA). 2001. Methods to Reduce/Eliminate Pathogens from Produce and Fresh-Cut Produce. In: Analysis and Evaluation of Preventive Control Measures for the Control and Reduction/Elimination of Microbial Hazards on Fresh and Fresh-Cut Produce. Accessed on 12/14/11. Available at: http://www.fda.gov/Food/ScienceResearch/ResearchAreas/SafePracticesforFoodProcesse s/ucm091363.htm
Food and Drug Administration (FDA). 2009a. Report on the occurrence of foodborne illness risk factors in selected institutional foodservice, restaurant, and retail food store facility types. U.S. Food and Drug Administration, Silver Spring, MD.
Food and Drug Administration (FDA). 2009b. Food Code, pp. 21 and 130. U.S. Food and Drug Administration, Silver Spring, MD.
Gerba, CP. 2009. Disinfection. In: Maier, RM, Pepper, IL and Gerba, CP (eds.). Environmental Microbiology, 2nd ed. Academic Press. San Diego, CA. pp. 539-552.
Gibson, KE and Schwab, KJ. 2011. Thermal inactivation of human norovirus surrogates. Food and Environmental Virology, 3:74-77.
Goodgame, R. 2007. Norovirus gastroenteritis. Current Infectious Disease Reports, 9:102–109.
Gulati, BR, Allwood, PB, Hedberg, CW and Goyal, SM. 2001. Efficacy of commonly used disinfectants for the inactivation of calicivirus on strawberry, lettuce, and a food- contact surface. Journal of Food Protection, 64:1430-1434.
Hall, AJ, Vinjé, J, Lopman, B, Park, GW, Yen, C, Gregoricus, N and Parashar, U. 2011. Updated norovirus outbreak management and disease prevention guidelines. Morbidity and Mortality Weekly Report (MMWR) - Recommendations and Report, 60:1-15.
Handojo, A, Lee, J, Hipp, J, and Pascall, MA. 2009. Efficacy of electrolyzed water and an acidic formulation compared with regularly used chemical sanitizers for tableware 107 sanitization during mechanical and manual ware-washing protocols. Journal of Food Protection, 72:1315-1320.
Hedberg, CW, Smith, SJ, Kirkland, E, Radke, V, Jones, TF and Selman CA. 2006. Systematic environmental evaluations to identify food safety differences between outbreak and nonoutbreak restaurants. Journal of Food Protection, 69:2697-2702. Hegstad,K, Langsrud, S, Lunestad, BT, Scheie, AA, Sunde, M and Yazdankhah, SP. 2010. Does the wide use of quaternary ammonium compounds enhance the selection and spread of antimicrobial resistance and thus threaten our health? Microbial Drug Resistance, 16:91-104.
Hirneisen, KA, Black, EP, Cascarino, JL, Fino, VR, Hoover, DG and Kniel, KE. 2010. Viral inactivation in foods: A review of traditional and novel food-processing technologies. Comprehensive Reviews in Food Science and Food Safety, 9:3-20.
Ioannou, CJ, Hanlon, GW and Denyer, SP. 2007. Action of disinfectant quaternary ammonium compounds against Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, 71:296-306.
Jimenez, L and Chiang, M. 2006. Virucidal activity of a quaternary ammonium compound disinfectant against feline calicivirus: A surrogate for norovirus. American Journal of Infection Control, 34:269-273.
Koopmans, M. and Duizer, E. 2004. Foodborne viruses: An emerging problem. International Journal of Food Microbiology, 90:23-41.
Kuda, T, Yano, T and Kuda, MT. 2008. Resistances to benzalkonium chloride of bacteria dried with food elements on stainless steel surface. LWT- Food Science and Technology, 41:988-993.
Kusumaningrum, HD, van Putten, MM, Rombouts, FM and Beumer, RR. 2002. Effects of antibacterial dishwashing liquid on foodborne pathogens and competitive microorganisms in kitchen sponges. Journal of Food Protection, 65:61-65.
Kusumaningrum, HD, Riboldi, G, Hazeleger, WC, Beumer, RR. 2003. Survival of foodborne pathogens on stainless steel surfaces and cross-contamination to foods. International Journal of Food Microbiology, 85:227-36.
Lee, J, Cartwright, R, Grueser, T and Pascall, MA. 2007. Efficiency of manual dishwashing conditions on bacterial survival on eating utensils. Journal of Food Engineering, 80:885-891.
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Line, JE, Fain, AR, Moran, AB, Martin, LM, Lechowich, RV, Carosella, JM, and Brown, WL. 1991. Lethality of heat to Escherichia coli O157:H7: D-value and Z-value determinations in ground beef. Journal of Food Protection, 54:762-766.
Lou, F, Neeto, H, Chen, H and Li, J. 2011. Inactivation of human norovirus surrogate by high-pressure processing: Effectiveness, mechanism, and potential application in the fresh produce industry. Applied and Environmental Microbiology, 77:1862-1871.
Malik, YS and Goyal, SM. 2006. Virucidal efficacy of sodium bicarbonate on a food contact surface against feline calicivirus, a norovirus surrogate. International Journal of Food Microbiology, 109:160-163.
Moe, CL. 2009. Preventing norovirus transmission: How should we handle food handlers? Clinical Infectious Diseases, 48:38-40.
National Restaurant Association (NRA). 2010. Restaurant Industry Forecast (2010).
Nowak, P, Topping, JR, Fotheringham, V, Gallimore, CI, Gray, JJ, Iturriza-Gómara, M and Knight, AI. 2011. Measurement of the virolysis of human GII.4 norovirus in response to disinfectants and sanitisers. Journal of Virological Methods, 174:7-11.
Rutala, WA and Weber, DJ. Uses of inorganic hypochlorite (bleach) in health-care facilities, Clinical Microbiology Reviews, 10:597-610.
Sanglay, GC, Li, J, Uribe, RM and Lee, K. 2011. Electron-beam inactivation of norovirus surrogate in fresh produce and model systems. Journal of Food Protection, 74:1155-1160.
Sigua, G, Lee, Y-H, Lee, J, Lee, K, Hipp, J and Pascall, MA. 2011. Comparative efficacies of various chemical sanitizers for warewashing operations in restaurants. Food Control, 22:13-19.
Springthorpe, VS and Sattar, SA. 1990. Chemical disinfection of virus-contaminated surfaces. Critical Reviews in Environmental Control, 20:169-229.
Stewart, MH and Olson, BH. 1996. Bacterial resistance to potable water disinfectants. In: Hurst, CH (ed.). Modeling Disease Transmission and Its Prevention by Disinfection. Cambrige University Press. Cambridge, UK. pp. 140-192.
Takahashi, H, Ohuchi, A, Miya, S, Izawa, Y and Kimura, B. 2011. Effect of food residues on norovirus survival on stainless steel surfaces. PLoS ONE, 6:e21951. doi:10.1371/journal.pone.0021951
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Verhoef, LP, Kroneman, A, van Duynhoven, Y, Boshuizen, H, van Pelt, W and Koopmans, M. 2009. Selection tool for foodborne norovirus outbreaks. Emerging Infectious Diseases, 15:31-38.
Whitehead, K and McCue, KA. 2010. Virucidal efficacy of disinfectant actives against feline calicivirus, a surrogate for norovirus, in a short contact time. American Journal of Infection Control, 38:26-30.
Wobus, CE, Thackaray, LB and Virgin IV, HW. 2006. Murine norovirus: A model system to study norovirus biology and pathogenesis. Journal of Virology, 80:5104-5112.
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CHAPTER 3: EFFICACY OF WARE-WASHING PROTOCOLS FOR THE
REMOVAL OF CALICIVIRUSES FROM FOOD CONTACT SURFACES
3.1. Abstract
The contamination of food contact surfaces with caliciviruses (e.g. noroviruses and sapoviruses) could present a major health risk if these surfaces are not effectively washed and sanitized. Therefore, the objective of this study was to evaluate the sanitization efficacies of common ware-washing protocols (mechanical and manual) for the removal of dairy products (contaminated with porcine sapovirus) from tableware items (ceramic plates, drinking glasses and stainless steel forks). The contaminated tableware items were washed and subsequently sanitized with different sanitizing solutions [sodium hypochlorite (chlorine), quaternary ammonium (QAC) and tap water
(control)]. Results showed that only the chlorine sanitizer was able to reduce PoSaV by approximately 2 logs when exposed to higher temperatures during mechanical ware- washing (49ºC vs. 43ºC during manual ware-washing). The other sanitizers did not produce significant reductions in the viral numbers (< 1 log). This study revealed that current ware-washing protocols and common sanitizing solutions are not effective against
111 non-enveloped viruses. Therefore, further studies are required to determine more effective methods for the removal and inactivation of caliciviruses from food contact surfaces.
3.2. Introduction
Human caliciviruses are responsible for the majority of the foodborne illness diseases in the United States (Scallan et al., 2011; Monroe et al., 2000). The inability to propagate caliciviruses (e.g. noroviruses and sapoviruses) in cell culture systems is a major obstacle during virus inactivation studies that seek to find better prevention and control of these viruses. Therefore, suitable and culturable surrogates able to resemble gastroenteritis symptoms caused by caliciviruses are essential. Most viral inactivation studies are focused on noroviruses (NoVs), which is responsible for 58% of all the foodborne illness cases (Scallan et al., 2011). Murine norovirus (MNV-1) has been used as a surrogate during NoV studies because of its genetic relatedness and its ability to survive under different stressors, including acidic conditions and high temperatures
(Taube et al. 2009; Cannon et al. 2006; Wobus et al. 2006). Also, MNV-1 is the only
NoV that replicates in cell culture and in small animal models (Wobus et al., 2004; Karst et al., 2003). However, MNV-1 does not cause the common symptoms of NoV infection
(e.g. vomiting and diarrhea) but it can be disseminated to multiple peripheral tissues and cause lethal systemic diseases (Mumphrey et al., 2007; Wobus et al., 2006). Porcine sapovirus (PoSaV) is a culturable calicivirus that resembles gastroenteritis infections caused by NoVs and sapoviruses (SaVs) (Chang et al., 2004; Chiba et al., 2000). Also,
112
PoSaV have shown to be resistant to low pH, high temperatures, and chlorine solutions, which makes it a potential surrogate for studying human caliciviruses (Wang et al.,
2012). This is so because it will act as a worst case scenario during challenge studies involving inactivation studies.
Food can be contaminated anywhere from farm to table. Today, restaurants and other food service establishments play an important role in the contamination of foods, especially ready-to eat (RTE) foods (Hedberg et al., 2006). Even though RTE foods are generally contaminated during the preparation or serving time by an infected food- handler (Ozawa et al., 2007), they can also become contaminated by food contact surfaces not properly cleaned or sanitized (Wernersson et al., 2004; Cogan et al., 2002).
Improper cleaning and sanitizing of food contact surfaces (e.g. tableware items) remains a serious problem in food service establishments (FDA 2009a; 2004; 2000). This can support the assumption that tableware items that are not properly washed and sanitized could potentially become a source for the transmission of caliciviruses. As a result, this study evaluated the sanitization efficacies of ware-washing protocols and common sanitizers (sodium hypochlorite and QAC) used in restaurants to remove PoSaV from different contaminated tableware items. This virus was used as a surrogate for human caliciviruses.
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3.3. Materials and Methods
3.3.1 Cell culture and virus stock
The porcine enteric calicivirus (PEC), strain Cowden, and the porcine kidney cell line LLC-PK, were provided by Dr. Linda Saif from the Ohio Agricultural Research and
Development Center (OARDC) located at the OSU Wooster Campus. The preparation and infectious titer assays of PoSaV were performed using porcine kidney cell line LLC-
PK. The cells were cultured and maintained in tissue culture flasks (150 cm2; BD Falcon,
Bedford, MA) containing growth medium [Minimum Essential Medium (MEM; GIBCO-
Invitrogen, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS; HyClone,
Logan, UT), 1% MEM Non-Essential Amino Acid (NEAA; GIBCO-Invitrogen, Grand
Island, NY) and 1% Antibiotic-Antimycotic (Anti-Anti; GIBCO-Invitrogen, Grand
Island, NY)] at 37ºC and 5% CO2 atmosphere. In order to infect the LLC-PK cells, the growth medium was removed and replaced with medium containing only MEM, 1%
NEAA and 1% Anti-Anti (Maintenance medium). The cells were incubated for 2-3 h prior to infection at 37ºC and 5% CO2. After incubation, the medium was discarded and the LLC-PK cells were infected with PoSaV at a multiplicity of infection (MOI) of 0.01.
The flasks containing the cells were then incubated at 37ºC and 5% CO2 for 1 h, with agitation every 15 min. After 1 h incubation, maintenance medium containing 50 μM of glycochenodeoxycholic acid sodium salt (GCDCA; Sigma, St. Louis, MO) was added to the flask and it was then incubated at 37ºC and 5% CO2 for 96 h. The virus was harvested by freeze-thawing three times at -80ºC and 37ºC, respectively. PoSaV was then purified by transferring the cell-virus suspensions into 50 ml sterile conical centrifuge tubes (USA
114
Scientific, Ocala, FL) and centrifugation at 3,000 rpm for 20 min using an Allegra 6R centrifuge with a GH-3.8 swinging bucket rotor (Beckman Coulter, Brea, CA). The supernatant was collected and stored at 4ºC for immediate use or at -80ºC in aliquots for future use.
3.3.2. Food samples preparation and inoculation
Cream cheese spread and 2% reduced fat UHT milk were used to contaminate the food contact surfaces in this study. The food items were purchased from a local grocery store in Columbus, OH. They were stored in a refrigerator at 4ºC until ready to use.
Ceramic plates, stainless steel forks and drinking glasses were the tableware items tested.
These were sterilized by autoclaving at 121ºC for 20 min before each experiment.
3.3.2.1. Ceramic plates
For contamination of the ceramic plates, 90 g of cream cheese were weighed in a sterile beaker, heated for 10 sec in a microwave, inoculated with 10 ml of the virus stock
(1:10 w/w) to provide a final titer of approximately 7 log plaques forming units per gram
(PFU/g). This was then stirred with a sterile tongue depressor (Fisher Scientific,
Florence, KY) to ensure proper mixing of the virus with the cream cheese. A total of 3 g of this cream cheese spread was applied to the entire food contact surface of each ceramic plate. The plates were air dried for 1 h at room temperature (25ºC) on a flat, sterile rack prior to the washing protocol.
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3.3.2.2. Forks and drinking glasses
A 0.5 g aliquot of the contaminated cream cheese (see section 3.3.2.1) was applied to each fork. For contamination of the glasses, 45 ml of milk were transferred to a
50 ml sterile conical tube and inoculated with 5 ml of the virus stock (1:10 v/v). From this solution, 0.5 ml was applied to the inner wall of each drinking glass. After contamination, both the forks and the glasses were then air dried for 1 h at room temperature (25ºC) on a flat, sterile rack prior to the washing protocol.
3.3.3. Preparation of the detergents and sanitizing solutions
Two different detergents were used during the washing step. Ecotemp Ultra Klene detergent (Ecolab, Inc., St. Paul, MN) was used for the mechanical washing, whereas
Monsoon detergent (Ecolab, Inc., St. Paul, MN) was used for the manual washing. The
Ultra Klene detergent was used at 3,000 ppm concentration and its ingredients included sodium hydroxide, sodium polyacrylate and sodium phosphonobutanetricarboxylate. The concentration used for the Monsoon detergent was 100 ppm and this detergent contained linear alcohol (C12-C15), decylamine oxide, linear alcochol (C9-C11) and lauryl dimethylamine oxide as active ingredients. These concentrations were within the range recommended by the manufacturer, as per the Food Code requirements.
The two sanitizers used during this study were sodium hypochlorite (chlorine- bleach) and quaternary ammonium compounds (QAC). The chlorine bleach contained
6% sodium hypochlorite and was purchased from a local grocery store in the Columbus,
OH area. The sodium hypochlorite solution used in this study was 200 ± 20 ppm. This
116 concentration was determined using a HI 95771 Clorine Ultra High Range Meter (Hanna
Instruments, Ann Arbor, MI). The QAC was an OASIS 146 Multi-Quat sanitizer, manufactured by Ecolab, Inc. (St. Paul, MN). The active ingredients found in this sanitizer were n-Alkyl (50% C14, 40% C12, 10% C16), dimethyl benzyl ammonium chloride (3.000%), octyl decyl dimethyl ammonium chloride (2.250%), didecyl dimethyl ammonium chloride (1.125%) and dioctyl dimethyl ammonium chloride (1.125%). This
QAC solution was used at a concentration of 200 ppm as determined by a HYDRION
QT-10 Quat test paper (QA Supplies, Norfolk, VA). According to the FDA Code
(2009b), QAC sanitizers must be mixed with water with a hardness level less than 500 ppm CaCO3. To ensure compliance with this, the water hardness was determined using a
Water Quality Test Strip kit (Hach Co., Loveland, CO). The water hardness for both mechanical and manual washing procedures was determined to be less than 120 ppm.
3.3.4. Mechanical ware-washing and sanitization of contaminated tableware items
For the mechanical ware-washing study, a LXiC Dishwasher manufactured by
Hobart Corp. (Troy, OH) was used. This machine was connected to a hot water line and had an incoming water pressure of 138 kPa. Prior to each experiment, the machine was cleaned with hot water (49°C) and filled with fresh water. The dishwasher water tank held approximately 11.4 liter. To ensure the proper amount of detergent during the washing cycle, the detergent (Ecotemp Ultra Klene detergent) was directly added to the water tank. The resulting water-detergent solution had a concentration of 3,000 ppm
(v/v). During the washing cycle, the tableware items were automatically sprayed with the
117 wash water for 76.5 s at a pressure and temperature of 138 kPa and 49°C, respectively.
After the washing cycle, the tableware items were automatically sprayed with the chlorine solution for 10 s at 49°C. Once the sanitizing cycle was completed, all tableware items were air dried for 1 h at 24 ± 2°C. This entire cycle was repeated but with the control and QAC solutions instead.
3.3.5. Manual ware-washing and sanitization of contaminated tableware items
For the manual dishwasher, a three compartment sink, manufactured by Eagle
Group, Inc. (Clayton, DE), was used for the washing, rinsing and sanitizing of the tableware items. Prior to each experimental run, the entire sink was thoroughly cleaned with hot water and refilled with fresh water and detergent/sanitizer. Each of the three compartments was filled with 28 liters of water. The tableware items were washed with
100 ppm of the Monsoon detergent at 43°C for 30 s, rinsed with tap water at 24°C for 10 s, and sanitized with one of the sanitizing treatments at 24°C for 30 s. The test was repeated for each sanitizer and tableware item. Rubber gloves were worn by the laboratory technician who washed the tableware items throughout the experiment.
During the washing step, each ceramic plate and fork was manually washed using a Scotch-Brite multi-purpose scrub sponge (3M, St. Paul, MN). To ensure consistency of the force applied to remove the cream cheese from the plates and the forks during the washing, the sponge was attached to a spring-loaded device. This allowed the technician to control the rubbing force applied to each tableware item. A cylindrical device covered with a soft sponge was used to wash the drinking glasses. The forks were washed using
118 fifteen forward and fifteen backward strokes with the sponge. The plates and glasses were washed using fifteen clockwise and fifteen counterclockwise strokes. After washing, the tableware were rinsed, sanitized and placed in a clean rack and air dried for 1 h at 24 ±
2°C. The three compartments of the sink were washed with hot water (49ºC) and bleach
(10%) after each experimental run.
3.3.6. Viral sampling of the tableware surfaces: PoSaV 50% infective dose assay
The initial viral titer of the virus stock, as well as from the contaminated milk, was determined by transferring 200 μl from each to a test tube containing 1.8 ml of phosphate buffered saline (PBS). The titer of the contaminated cream cheese was determined by transferring a small amount of the cheese to a test tube containing 2 ml of
PBS using a sterile calcium-alginate cotton-tipped swab (Fisher Scientific, Pittsburgh,
PA). Prior to the ware-washing, 4 samples of each tableware item were collected after the air drying period. Once the sanitization step was completed and the tableware items air dried for 1 h, 4 samples of each item were collected. These samples (before and after the ware-washing procedure) were collected using cotton-tipped swabs moistened with the
PBS solution. The test tubes containing the samples were vortexed to remove any virus attached to the tip of the swab.
Serial dilutions (1:4) were performed in order to infect the LLC-PK cells monolayers. The viral samples were diluted (from 1:4, 4-fold serially dilution) in a sterile
96-well plates as followed: a 400 μl aliquot of sample for each dilution was prepared (100
μl of viral sample + 300 μl of the maintenance medium). To determine the initial viral
119 titer of the virus stock and of the contaminated milk, 100 μl of each were transferred directly to the sterile 96-well plate containing 300 μl of the maintenance medium. These were serially diluted as previously described. LLC-PK cells were previously grown in 96- well plates (1:3 passage, 200 μl/well). Cells were at least 3-day old, having a good monolayer with cell domes formed. Old growth medium was replaced with fresh maintenance medium (100 μl/well) and incubated for 2-3 h before virus inoculation.
After the incubation period, the medium from the 96-well plates with cell monolayer was removed and 50 µl of maintenance medium containing 2x concentration of GCDCA (100
µM) were added to each well. Then, 50 µl of the viral samples (per well) were transferred to the 96-well plates containing the monolayers (four replicates for each dilution). The inoculated plates were incubated at 37 ºC for 72 h and the cytopathic effect observed under the microscope. The virus titer was determined using a method described by Reed and Muench (1938).
3.3.7. Statistical Analysis
All experiments were repeated twice in this study. The viral counts were expressed as log PFU per tableware (surface). During each test, 4 tableware items were selected for viral quantification before and after sanitization. The reported values of the viral counts were the mean values of two trials ± standard deviations. Multifactor analysis of variance (ANOVA) was used to determine the significance between the mean values.
The data analyses were performed by the General Linear Model function and Tukey-
Kramer’s multiple comparison test using a SAS, version 9.2, statistical program (SAS
120
Institute, Cary, NC). This program was used to determine the significance between the effect of each sanitizer, tableware item and ware-washing protocol (manual vs. mechanical). A p value of 0.0001 was set for the level of significance.
3.4. Results and Discussion
3.4.1. Effect of air-drying on the reduction of PoSaV on the contaminated tableware
To ensure adherence of the contaminated food to the surfaces and simulate the average time between the use and cleaning of tableware items, they were allowed to air- dry for 1 h. The effect of this drying period on the PoSaV counts is presented in Figure
3.1. The mean reductions in the viral counts ranged between 0 to 0.2 log10 PFU per tableware item. These virus reductions on the three surfaces (plates, forks and drinking glasses) were relatively small and not significant (p > 0.0001).
Within recent times, the environmental stability of NoV on different surfaces and during drying conditions had been investigated (Takahashi et al., 2011; Cannon et al.,
2006; D’Souza et al., 2006). However, limited information can be found in the literature about the stability of SaVs in the environment or on surfaces exposed to drying conditions. Results from our study reveal that PoSaV is quite resistant to desiccation conditions, which suggests that if food contact surfaces are contaminated with SaV, these may become a source of contamination if the cleaning and sanitization practices fail to remove the infectious virus.
121
Plates Forks Glasses
9.0
8.0
7.0
6.0
5.0 PFU/tableware
10 4.0
Log 3.0
2.0
1.0
0.0 Pre-Drying Post- Drying
Figure 3.1. Survival of PoSaV on different contaminated tableware before and after 1 hour air-drying at 24 ± 2°C.
3.4.2. Comparison between the efficacies of the mechanical and manual ware- washing protocols and the effect of the sanitizers tested
Figure 3.2 shows the survivability of PoSaV on the contaminated surfaces before and after mechanical ware-washing. The control and the QAC sanitizers appear to have little effect on PoSaV since the viral counts after the ware-washing operation were not statistically different from the initial viral counts (pre-wash). The mean reductions of
PoSaV on the plates, forks and drinking glasses after washing treatment with the control were 0.9, 0.4 and 0.3 log, respectively. The mean reductions achieved after washing and
QAC sanitation (1.0, 0 and 0.1 log, respectively) were similar to those obtained by the 122 control treatment. On the contrary, the chlorine sanitizer appeared to work better than both the control and the QAC sanitizers. The mean reductions achieved after washing and chlorine sanitation were 2.2, 1.7 and 1.3 log on the plates, forks and drinking glasses, respectively. Statistically, these chlorine sanitizer reductions were significantly (p <
0.0001) higher when compared with the control and the QAC sanitizer.
Plates Forks Glasses 9.0
8.0
7.0
6.0
5.0
4.0 PFU/ tableware PFU/
10 3.0
Log 2.0
1.0
0.0 Pre-Wash Control Chlorine QAC Pre-Wash Control Chlorine QAC Mechanical Manual
Figure 3.2. Survival of PoSaV on contaminated tableware items after washing and sanitizing, using the mechanical and manual dishwasher, respectively.
123
The effect of manual ware-washing and sanitizing solutions on the reduction of
PoSaV from the contaminated tableware items are also presented in Figure 3.2. For the control treatment, the reductions ranged from 0 to 0.2 log per tableware item. The reductions achieved after washing and chlorine sanitation ranged from 0.1 to 1.1 log per tableware item. For the QAC sanitizer, the reductions ranged from 0.2 to 0.8 log per tableware item. In general, the mean manual washing reductions of PoSaV on the tableware items were lower than the reductions achieved after mechanical ware-washing.
No significant differences (p > 0.0001) were found between the reductions achieved after washing and sanitizing with the three sanitizers during manual ware-washing. However, significant differences (p < 0.0001) were found when the mean reductions of all the treatments during manual ware-washing were compared with the mean reductions obtained after mechanical ware-washing with the chlorine sanitizer.
Results presented in Figure 3.2 show the different response of PoSaV during the two ware-washing operations and exposure to different sanitizing solutions. The mechanical ware-washing in combination with the chlorine sanitizer seem to better enhance the removal/inactivation of PoSaV on contaminated tableware items. In order to understand the results from this study, several factors will be mentioned and discussed.
These include: 1) the nature of the virus (enveloped vs. non-enveloped); 2) characteristics of the sanitizing agents and their concentrations; 3) differences in the ware-washing methods; and 4) the type of food matrix used to soil the tableware items.
Generally, enveloped viruses are more susceptible to environmental stresses (e.g. dehydration, detergents, surfactants and disinfectants; Dvorakova et al., 2008; Lombardi
124 et al., 2008; Sattar and Springthorpe, 2001; ARMCANZ, 2000; McDonell and Russell,
1999) than non-enveloped viruses (e.g. PoSaV). This susceptibility has been attributed to the presence of essential lipids in the envelope of these viruses (Sattar, 2007; Klein and
Deforest, 1983). The envelope facilitates entry of the virus into host cells and replication of the virus. The envelope also acts as a protective shell for the fragile nucleic acids of the virus (Chan et al., 2010). Thus, if this envelope is damaged the virus becomes susceptible to injury and its ability to cause infection is impaired (McDonnell and Burke,
2011). The resistance of non-enveloped viruses to environmental stressors is attributed to their capsid composition, which is predominantly protein in nature (McDonnell and
Burke, 2011; McDonnell and Russell, 1999). Non-enveloped viruses only lose their ability to cause infection if the viral capsid proteins are damaged, and this prevents them from attaching to the receptor sites on the host cell or from releasing their genome into the host cell’s environment (Nwachcuku and Gerba, 2004; Thurman and Gerba, 1988).
However, viral inactivation would only be complete if the viral nucleic acid is also destroyed (McDonell and Russel, 1999).
To ensure inactivation of non-enveloped viruses, the proper sanitizer or chemical agent has to be selected. Unfortunately, the majority of the sanitizers used today in the food industry possess antimicrobial effect against a broad range of microorganisms but not against non-enveloped viruses. For instance, at high concentrations, QACs have shown to be lethal to vegetative bacteria, yeasts, mold, algae, and enveloped viruses, but not to bacterial spores, mycobacteria, or non-enveloped viruses (Hegstad et al., 2010;
Jimenez and Chiang, 2006; Eleraky et al., 2002; Merianos, 2001; McDonnell and Russell,
125
1999; Fredell, 1994). In this present study, the QAC sanitizer had little effect on the
PoSaV, and as a result, a considerable amount of the virus remained on the contaminated food contact surfaces. This is in agreement with what has been reported in the literature, where QAC has shown to be ineffective against non-enveloped viruses (Jimenez and
Chiang, 2006; Eleraky et al., 2002; Kennedy et al., 1995). The antimicrobial activity of
QACs has been mainly associated with their cationic surfactant properties (Kügler et al.,
2005; Thome et al., 2003; Fredell, 1994). When used to treat enveloped viruses, the mechanism of action of QAC is attributed to the disruption or removal of the envelope and/or micelle formation within the viral particles (Shirai et al., 2000).
Sodium hypochlorite is known for its oxidizing effect on a broad range of pathogens, including enveloped viruses (Peng et al., 2002; Rutala and Weber, 1997). The antimicrobial activity of sodium hypochlorite is most likely caused by the penetration of the chemical and its oxidative action on essential enzymes in microbial cells (Lomander et al., 2004). The sanitizing action of sodium hypochlorite is pH-dependent, given that its active species is undissociated hypochlorous acid (HOCl) (Rutala and Weber, 1997).
Sodium hypochlorite solutions are more effective when used at pH 4 to 7, because at this pH range HOCl is dominant (Christensen et al., 2008; McDonnell and Russel, 1999;
Rutala and Weber, 1997). As the pH increases, more hypochlorite ions (OCl-) are formed and the microbicidal activity decreases (Rutala and Weber, 1997; Springthorpe and
Sattar, 1990).
Several studies have evaluated the efficiency of sodium hypochlorite in inactivating non-enveloped viruses (Eterpi et al., 2009; Gulati et al., 2001; Doultree et al.,
126
1999). The findings of these previous studies have shown that non-enveloped viruses are quite resistant to the killing action of sodium hypochlorite, even at chlorine concentrations much higher than allowed by the FDA Code of Federal Regulations (21
CFR 178.1010). Even though SaVs are different than NoVs, they have been used as surrogates for NoV (Vashist et al., 2009; Cannon et al., 2006; Guo et al., 1999). A recent study conducted by Wang et al. (2012) showed that PoSaV and human NoV have similar resistance to heat and to different concentrations of chlorine, indicating that PoSaV is a promising surrogate for NoV studies. Thus, the results from this study could be used to complement and support the findings of Wang et al. (2012), that PoSaV is slightly affected after chlorine treatment. This can be associated with the fact that the pH of this present study was ≥ 7.5, and thus the main chlorine species present in the solution was in the form of OCl-. Ideally, to maximize the sanitizing capabilities of the solution, it would have been necessary to reduce the pH in order to increase the amount of HOCl. However, pH values between 6.0 and 7.5 are typically used in sanitizer solutions to minimize corrosion of the equipment while yielding acceptable chlorine efficacy (FDA, 2001).
In general, higher reductions of the PoSaV on the tableware items were observed during the mechanical ware-washing when chlorine was used as the sanitizing solution.
The main difference between these two ware-washing protocols is the temperature. The temperatures used for both the washing and the sanitizing cycles during the mechanical ware-washing were higher than those used during the manual ware-washing. The literature reports that the inactivation activity of biocides, including hypochlorites, is enhanced at high temperatures (Xu et al., 2008; Sirtes et al., 2005; Erkmen, 2004; Evanov
127 et al., 2004; Rutala and Weber, 1997; Dyshdala, 1991). Although hypochlorites were thought to deteriorate at high temperatures (Cunningham and Balekjian, 1980), more recent findings suggest that increasing the temperature of sodium hypochlorite solutions to at least 50ºC has no adverse effect on the chemicals’ stability (Sirtes et al., 2005;
Gambarini et al., 1998). Hence, the enhanced removal of PoSaV from the tableware items after sanitization with the chlorine treatment can be attributed to the higher temperature of this solution during the mechanical ware-washing exercise. Besides, since commercial products of sodium hypochlorite (i.e. cleaning bleaches) also include surfactants to improve hard-surface cleaning (Clarkson et al., 2006; Rutala and Weber, 1997), this could also enhance the removal of viruses at elevated temperatures.
In addition to what has been previously discussed, the type of food used is another important factor to be considered in explaining the results obtained in this study. Milk and milk-based products have been described as foods that are difficult to remove from tableware items (Handojo et al., 2009; Lee et al., 2007). This is so because of their relatively high fat and protein content which help to increase their adhesion to surfaces
(Schmidt, 2009; Sigua et al., 2009). This would help to reduce the effectiveness of the sanitizers. Although the tableware items tested in this study appeared visibly clean, a microscopic food layer may have formed while the food was allowed to adhere to the tableware items prior to the ware-washing protocol. This layer could have acted to protect the virus left within its matrix (Kusumaningrum et al. (2003). Therefore, even if the foods appeared to be removed during the washing cycle, the virus could have been tightly
128 attached to the surfaces and this could have affected the performance of the sanitizing solutions. Hence, further studies are required to confirm this.
3.5 Conclusions
Results from this study demonstrated that current ware-washing protocols and common sanitizing solutions used in foodservice establishments are not capable of removing non-enveloped viruses from contaminated food contact surfaces at FDA recommended conditions. Thus, there is a need for alternatives to hypochlorites and
QACs sanitizers. Also, there is a need to improve the current ware-washing protocols in an attempt to help control foodborne infections associated with viral contamination at restaurants and other foodservice settings.
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CHAPTER 4: THE USE OF ATOMIC FORCE MICROSCOPY TO COMPARE
THE EFFICACY OF DIFFERENT CHEMICAL SANITIZERS IN THE
REMOVAL OF MILK CONTAMINATED WITH VIRUSES ON GLASS AND
CERAMIC SURFACES
4.1. Abstract
Foodborne viruses are mainly composed of protein residues. Protein-based soils can strongly adhere to food contact surfaces and become a public health hazard if they are not properly removed during ware-washing protocols. This study investigated the ability of various sanitizing solutions such as chlorine-based sanitizers (bleach and Chlor-
Clean), quaternary ammonium compound (QAC) and tap water for the removal of milk samples from ceramic and glass surfaces. Two types of milk samples were tested. One type was contaminated with viruses and the other was not. Murine norovirus (MNV-1) and porcine sapovirus (PoSaV) were used as surrogates for human norovirus (NoV) and sapovirus (SaV). Atomic force microscopy (AFM) was used to determine the thicknesses of the milk films left after the surfaces were mechanically washed and sanitized. Results showed that the surfaces contaminated with the milk-viruses samples generally had thicker films when compared with the milk samples that did not contain the viruses. This could be due to a greater content of protein residues present in these milk samples. The data also showed that the sanitizing solutions may have affected the residual film thick- 136 nesses of the milk samples contaminated with MNV-1 and PoSaV. Further studies are needed to determine the optimum conditions for the removal of these viruses, including the proper selection of detergent and sanitizing solutions and to determine factors that may enhance or reduce the adhesion of viruses to food contact surfaces.
4.2. Introduction
Food contact surfaces may present a public health risk if they are not properly washed and sanitized. Residual food soils left on food contact surfaces could harbor pathogenic bacteria and viruses. Thus, foods such as those considered ready-to-eat (RTE) could then become contaminated when in contact with these soiled surfaces and subsequently help to disseminate pathogens known as important foodborne disease agents. To minimize this risk, the FDA Food Code (2009) and the ANSI/NSF
International standards (2009) mandate a 5 log bacterial reduction and the removal of all visible sign of food soils from tableware items and other food contact surfaces during ware-washing protocols.
Previous ware-washing studies have shown that milk-based products are difficult to remove from glass surfaces (Lee et al., 2007) and that their adhesion strengths
(residual film thickness) to these surfaces can be estimated using an AFM technique
(Sigua et al., 2010; Handojo et al., 2009). However, information regarding the influence different microorganisms may have in enhancing the adhesion of food soils to contact surfaces is quite limited. Caliciviruses are responsible for the majority of foodborne outbreaks that cause gastroenteritis. Noroviruses (NoVs) and sapoviruses (SaVs) are two
137 types of caliviruses that have been identified as causing acute gastroenteritis in humans
(Scallan et al., 2011; Tan and Jiang, 2010; Shirato-Horikoshi et al., 2007). However, current ware-washing protocol guidelines are based on studies designed for the reduction of bacterial populations, but not viruses. Studies addressing the persistence of non- enveloped viruses on food contact surfaces and their removal from these surfaces after ware-washing protocols are limited. Hence, the objective of this study was to use an
AFM technique to compare the ability of several sanitizing solutions to remove milk residues containing viruses from glass and ceramic surfaces after mechanical ware- washing protocols. Murine norovirus (MNV-1) and porcine sapovirus (PoSaV) were used as surrogates for NoV and SaV, respectively.
4.3. Materials and Methods
4.3.1. Food sample preparation and contact surfaces
Ultra high temperature (UHT) 2% reduced fat milk was purchased from a local grocery store (Columbus, OH) to contaminate glass and ceramic surfaces. This was done for the purpose of measuring the ability of AFM to test the effectiveness of the ware- washing protocols to remove residual milk from the simulated food contact surfaces. Pre- cleaned glass microscope slides and square ceramic samples (2”x 2”) were used to simulate the surfaces of drinking glasses and ceramic plates, respectively. For the contamination of the surfaces, 10 μl of milk were placed on separate glass microscope slides or ceramic surfaces and air-dried for 1 hour at room temperature (25ºC). This was done to allow the milk samples to sufficiently adhere to the contact surfaces, as well as to
138 simulate normal foodservice operations. The same procedure was followed for the milk samples contaminated with the viruses (MNV-1 or PoSaV). In this case, 45 ml of milk were transferred to a 50 ml sterile conical tube and inoculated with 5 ml of the virus stock solution (1:10 v/v). Then, 10 μl of this solution were placed on the contact surfaces. After
1 hour drying, the glass microscope slides were attached to a thick ceramic tile
(previously cleaned with 70% ethanol solution) using tape at one end of the glass slides to prevent them from getting into the washing water tank (see Figure 4.1).
Figure 4.1. Illustration of milk samples on glass and ceramic surfaces inside the mechanical ware-washer.
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4.3.2. Mechanical ware-washing protocol
After the milk samples were prepared, they were mechanically washed in a water- detergent solution of 3,000 ppm of Ecotemp Ultra Klene detergent (Ecolab, Inc., St. Paul,
MN) at 49ºC for 76.5 s. After the washing cycle, the glass and ceramic surfaces were automatically sprayed with quaternary ammonium compound (QAC) sanitizing solution
(200 ppm) for 10 s at 49°C. Once the sanitizing cycle was completed, all the surfaces were air dried for 1 h at 24 ± 2°C. This entire cycle was repeated but with the sodium hypochlorite sanitizers (bleach and Chlor-Clean, respectively) instead. The chlorine concentration for these two solutions was 200 ppm. Tap water was used as the control sanitizing treatment. The ware-washing procedures used in this study simulated the mechanical ware-washing protocol required by the FDA Food Code (2009) for tableware items.
4.3.3. Atomic force microscopy (AFM) measurements
The AFM measurements were carried out using a Nanoscope III Dimension 3100 microscope (Veeco, Santa Barbara, CA) at room temperature. Contact and tapping modes were used to determine the thickness of residual milk samples left on glass and ceramic surfaces after the mechanical ware-washing procedure. The AFM nanoprobe cantilever was made of silicon (Si), was coated with platinum-iridium (PtIr) on both sides (front and back) and had a nominal spring constant ranging from 1-5 N/m. It was obtained from
Bruker Corp. (Camarillo, CA). The length of the cantilever was 200-250 μm. This type of cantilever was selected after several trials were made with an uncoated cantilever made
140 of Si, which was not able to completely remove the milk samples on the surfaces, especially those containing the viruses. Therefore, a trial with a Si-PtIr coated tip easily removed the milk films and proved to achieve better results.
During the contact mode, the AFM tip was used to remove a 25 x 25 μm area of each milk sample from the underlying glass and ceramic surfaces, respectively. The applied force during this mode was proportional to the AFM set point potential, which was the voltage measured by the photodiode detector. The set point was controlled at 11
V. This high voltage was selected after preliminary results showed it was adequate to completely remove milk residual films (e.g. milk samples containing viruses) from the surfaces. Therefore, the voltage was kept constant and each sample was scratched 15 times to ensure the complete removal of the milk residual films. Once scratched, the resultant surface was imaged in tapping mode (see Figure 4.2). The size of the image obtained in this mode was 75 x 75 μm. The thickness (depth) of the milk residual samples was determined from cross-sectional measurements through the scratched area, generated by the Nanoscope III computer software.
141
Figure 4.2. A tapping 3-D topographical AFM image of the surface a milk-MNV-1 sample on the glass surface after chlorine sanitization and the contact mode scratching test.
4.3.4. Statistical analysis
The reported results were averages of nine measurements ± standard errors.
Multifactor analysis of variance (ANOVA) was used to determine the significance between the mean values. The data analyses were performed by the General Linear
Model function and Tukey’s multiple comparison test with the SAS, version 9.2, statistical program (SAS Institute, Cary, NC) to determine the level of significance between the effect of each sanitizer, contact surface, and type of milk sample (with or without virus). To properly identify any significant differences at low levels, the p value was set at < 0.0001.
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4.4. Results and Discussion
4.4.1. AFM measurements of the milk film thickness on ceramic and glass surfaces after mechanical ware-washing
The average residual milk film thicknesses on the ceramic and the glass surfaces after the mechanical ware-washing are presented in Figure 4.3. For both the ceramic and the glass surfaces, four different types of samples were analyzed: 1) bare surface; 2) surface containing only milk; 3) surface containing milk contaminated with MNV-1; and
4) surface containing milk contaminated with PoSaV. Bare surfaces were analyzed in order to quantify the residual film left as a result of the sanitizing solutions. Results for the bare ceramic surface show that the QAC sanitizer produced a thicker film (12.6 nm) when compared to the other treatments. The mean residual film left on the ceramic surface after exposure to the control (tap water) sanitizer was 8.4 nm whereas the mean thicknesses after chlorine and Chlor-Clean sanitization were 9.1 and 6.3 nm, respectively.
Statistically, only the mean film thickness on the ceramic (after exposure to the QAC sanitizer) was significantly different (p < 0.0001) when compared with that of the other sanitizers. For the glass surfaces, no significant differences (p > 0.0001) were found among the thicknesses of all the sanitizing solutions’ residual films. The films produced after exposure to the control and the QAC solutions were relatively similar in thickness
(4.5 and 4.2 nm, respectively). On the other hand, the film thicknesses after chlorine (3.3 nm) and Chlor-clean (2.5 nm) sanitization were slightly lower than the control and QAC.
Interestingly, the ceramic surfaces that contain only milk had thinner residual films
(control (4.1 nm), QAC (6.4 nm), chlorine (2.9 nm) and Chlor-Clean (4.3 nm),
143 respectively) when compared to the films left on the bare ceramic surfaces. To determine if the difference observed between these two types of samples (bare ceramic vs. milk only) had an associated effect with the detergent solution used during the washing cycle, both the bare ceramic and glass surfaces were washed without the use of the detergent
(Figure 4.4). This procedure was done only with the control sanitizer. The results presented in Figure 4.4 show that the thickness of the films left on both surfaces (ceramic and glass) was reduced when the detergent was not included during the washing cycle. Of the two surfaces, the contribution of the detergent to the residual film thickness appeared to be higher on the ceramic than on the glass surfaces. This effect of the detergent was statistically significant (p < 0.0001) for both the ceramic and the glass surfaces.
Results in Figure 4.3 also show the effect of viruses on the adhesion of the milk to the ceramic and glass surfaces. For the ceramic contaminated with milk-MNV-1 sample, the results show that both the control and the Chlor-Clean sanitizers produced the highest thicknesses (17.1 and 17.7 nm, respectively), while the QAC and chlorine sanitizers caused the lowest thicknesses (10.8 and 10.9 nm, respectively). These differences in the mean thicknesses (control and Chlor-Clean vs. QAC and chlorine) were statistically significant (p < 0.0001). In the case of the glass surfaces contaminated with the milk-
MNV-1sample, only the chlorine sanitizer produced a significantly (p < 0.0001) thicker residual film (20.5 nm). The film thicknesses produced after exposure to the control (5.0 nm), QAC (7.4 nm) and Chlor-Clean (5.1 nm) sanitizers were not significantly different
(p > 0.0001) from each other. The thicknesses obtained for the milk-PoSaV samples were a little different than those obtained for the milk-MNV-1 samples. For example, the milk-
144
PoSaV samples on the ceramic surface sanitized with the control sanitizer had a thickness of 12.3 nm. This film thickness was significantly different from the film produced after chlorine sanitization (19.6 nm), but was not significantly different from the film produced after the QAC (15.5 nm) or the Chlor-Clean (13.7 nm) sanitization. Besides, the differences in film thicknesses after sanitization with the QAC and the chlorine sanitizers were statistically significant (p < 0.0001).
For the glass surfaces contaminated with the viruses, the residual film thickness results differed from those of the ceramic surfaces. For instance, the thickest milk-MNV-
1 film was found after sanitization with the chlorine sanitizer (20.5 nm). This thickness was significantly different (p < 0.0001) from the thicknesses produced with the other sanitizing solutions. The milk-MNV-1 film thicknesses for the control, QAC and Chlor-
Clean sanitizers were 5.0, 7.4 and 5.1 nm, respectively. No significant difference (p >
0.0001) was found among these results. For the milk-PoSaV samples, on the other hand, the film thicknesses differed from one sanitizer to another. The thickest milk-PoSaV film measured on the glass surfaces was that produced after QAC sanitization (25.0 nm), which was significantly higher (p < 0.0001) when compared with the films obtained with the rest of the sanitizing solutions. The film thickness produced after sanitization with the control was 2.9 nm and this was statistically different (p < 0.0001) from the thicknesses produced with the chlorine (9.1 nm) sanitizer. However, no significant differences (p >
0.0001) were found between the thicknesses measured for the control and the Chlor-
Clean sanitizers (4.5 nm). The milk-PoSaV film produced with the chlorine sanitizer was statistically thicker (p < 0.0001) than the Chlor-Clean resulting film.
145
Control QAC Chlorine (Bleach) Chlor-Clean 35.0
30.0
25.0
20.0
15.0 Thickness (nm) Thickness
10.0 Film
5.0
0.0 Bare Surface Milk Milk + MNV Milk + Bare surface Milk Milk + MNV Milk + PoSaV PoSaV CeramicCeramic GlassGlass
Figure 4.3. AFM mean thicknesses (nm) of milk samples on ceramic and glass surfaces.
12.0
Bare surface sanitized with control 10.0
8.0
6.0
Thickness (nm) Thickness 4.0
Film 2.0
0.0 No detergent during Detergent during No detergent during Detergent during washing washing washing washing
CeramicCeramic GlassGlass
Figure 4.4. Effect of the detergent solution on the film thickness left on bare ceramic and glass surfaces during the mechanical ware-washing protocol.
146
In general, results presented in Figure 4.3 show that the milk samples (no virus) on the contact surfaces had relatively thinner films when compared to the milk samples contaminated with the viruses. These results suggest that the presence of viral particles enhanced the adherence of the milk samples to the surfaces, especially to the ceramics.
There are several possible explanations that can help to understand the results obtained in our study. For example, non-enveloped viruses (e.g. MNV-1 and PoSaV) are composed mainly of proteins, which are made up of hydrophilic, hydrophobic, and neutral residues
(Casanova et al., 2009; Vega et al., 2008). The sum of all these residues/forces can influence the adhesion of viruses to surfaces (Vega et al., 2008). Electrostatic interactions, van der Waals forces, and hydrophobic effects are assumed to play a role in the interactions between virus particles and contact surfaces (Gibson et al., 2012;
Casanova et al., 2009; Gerba, 1984). However, electrostatic forces appear to govern the attachment of viruses to surfaces (Casanova et al., 2009). Also, electrostatic forces are believed to play a major role on protein adsorption at hydrophilic surfaces, only if the net charge of the proteins is opposite to the surface (Santos et al., 2004; Michalski et al.,
1997; Norde et al., 1991). Therefore, virus attachment to ceramic and glass surfaces may be attributed to electrostatic interactions, where charged viral surfaces (e.g. viral capsid) may encounter charged groups on these hydrophilic surfaces (Casanova et al., 2009). Yet, scientific data concerning surface properties of viruses and prediction of their adhesion capacity to different surfaces is limited (Deboosere et al., 2012).
Another possible explanation for the enhanced adherence of the viruses- contaminated milk samples to the contact surfaces is the surface energy of the surfaces
147 themselves. According to the literature, surfaces with high surface free energy (e.g. hydrophilic surfaces) generally show greater microbial attachment than hydrophobic surfaces. The reason for this is that microorganisms tend to attach uniformly in a monolayer to hydrophilic surfaces while on hydrophobic surfaces they tend to adhere in clumps (Mamvura et al., 2011; Chmielewski and Frank, 2003). However, opinions regarding this issue are still in controversy.
The milk-drying time before the ware-washing protocol may have also contributed to the high milk thicknesses observed in the surfaces contaminated with the viruses. It has been reported that when a protein attaches to an interface, it may undergo shape changes (van der Veen et al. 2007). These conformational changes are more pronounced as contact time with the interface has increased, where hydrophobic amino acids from the interior core of the protein have presumably moved to the surface where they can interact with the substrate (Xu and Siedlecki, 2007). Girard et al. (2010) observed a maximum attachment of MNV-1 to stainless steel surfaces (hydrophilic) after a contact time of 10 min, although an extension in the contact time to 60 or 120 min did not increase the extent of the attachment. Results from Girard et al. (2010) support the assumption that contact time facilitated the adhesion of milk-viruses samples to contact surfaces. It is important to mention that milk itself is composed of different dissolved solids. For example, the milk used in this present study was a 2% reduced fat UHT milk, which is mainly composed of water (89%), carbohydrates (5%), proteins (3%) and fats
(2%) (USDA, 2011). The high amount of water in these samples may have facilitated the adhesion of the milk-protein films to the surface. This is so because as contact time
148 increases, the water contained in the liquid will eventually evaporate and lead to the formation of solid bridges (e.g. chemical adhesion) between the residual film and the surface (Karbowiak, 2006; Adhikari et al., 2001; Michalski et al., 1997).
The effect of the sanitizing solutions on the thicknesses of the residual films, particularly on the glass surfaces, is another important factor that needs to be considered.
Even though MNV-1 and PoSaV showed different adhesion patterns (e.g. film thicknesses) to the contact surfaces, the residual films of these viruses were significantly higher after sanitization with the chlorine and QAC sanitizers, respectively. This can probably be attributed to differences in the interactions these viruses may have had with specific compounds in the sanitizing solutions. A study conducted by Tian et al. (2011) showed that the adhesion of human norovirus (NoV) to strawberry and lettuce surfaces was enhanced when these food items were washed with acidic electrolyzed water (AEW).
They attributed this “binding enhancement” to nonspecific ionic interactions between
NoV and the surfaces treated with AEW. Although the plant surfaces are relatively different from that of the contact surfaces in this study (hydrophobic vs. hydrophilic), results from Tian et al. (2011) study provide basic information that can help to support the results obtained in our study, where it is hypothesized that each virus had a specific interaction with the chlorine and QAC sanitizing solutions. However, further studies are needed to confirm this assumption. A study conducted by Sigua and collaborators (2010) showed that an organic acid sanitizer had a better ability to remove milk films adhered to glass surfaces when compared to chlorine and QAC sanitizers. The reason for this was the presence of a surfactant molecule (sodium dodecylbenzene sulfonate) in the acidic
149 formulation of the sanitizer. Therefore, the removal of milk-viruses films may be enhanced if an organic acid containing a surfactant agent is used.
The detergent effect observed on the residual film thicknesses left on the bare ceramic and glass surfaces (Figure 4.4) is an important factor that merits some potential explanations. The detergent used in this study contains sodium hydroxide as its main ingredient (23% by weight). It is reported that when hydrophilic surfaces (e.g. glass) are cleaned with sodium hydroxide solutions the surfaces readily become more hydrophilic.
Thus, the affinity of the surfaces for water molecules and the wettability properties increase (Harnett et al., 2007). Since detergent solutions contain both polar and non-polar groups, it could then be expected that as the hydrophilic properties of the contact surfaces increase, so too will be the interaction of the detergent-polar groups in aqueous media.
However, when food/milk soils are present on these contact surfaces, the hydrophobic groups of the detergent molecule are then associated to the hydrophobic groups of the food/milk soils (Horton et al., 2006; Friedman and Wolf, 1996), thus, enhancing the removal of excess detergent-soil- residues as seen in Figure 4.3.
Despite all the potential explanations given to interpret the variability observed for the film thicknesses, it is important to consider some possible errors. For example, the fact that the force used during this study was the maximum for the AFM machine (11v) and that the number of scans done during contact mode was fixed at 15 scans per sample, this may have limited the information provided about the amount of milk soils actually removed from the samples. This meant that the possibility that some residual milk might have been present on the contact surfaces. The association of surface roughness and
150 retention of soil and microorganisms can be found in the literature (Ayebah and Hung,
2005; Verran and Boyd, 2001; Korber et al., 1997). Therefore, further studies are needed to ensure that all the milk soils are completely removed from the food contact surfaces before completion of the test. To achieve this goal, the roughness of the bare ceramic and glass surfaces should be investigated and compared to the roughness of the scratch surfaces containing residual milk soils. This would give the analyst an idea of how completely the scratching removed the soils. Also, the degree of roughness and the effect of surface modification on the removal of milk samples should be investigated since these factors play an important role during cleaning procedures (Detry et al., 2010;
Saikhwan et al., 2006; Jullien et al., 2002).
4.5. Conclusion
This study demonstrated that an AFM technique can be used to determine the thickness of residual soils contaminated with viruses on food contact surfaces after ware- washing protocols. Also, this study suggested that milk soils contaminated with viruses tend to adhere to a greater extent (thicker films) to ceramic and glass surfaces than non- contaminated soils. Common sanitizing solutions (chlorine-based and QAC sanitizers) appeared not to effectively remove milk-virus deposits from simulated food contact surfaces. Therefore, foodborne viruses strongly adhered to food contact surfaces may be transferred to foods and become a source for the transmission of infectious viral particles even after washing in traditional washers and using traditional protocols. Therefore, future studies are needed to determine the optimum conditions for the removal of these
151 viruses, including the proper selection of detergent and sanitizing solutions. In addition to this, a comparison between hydrophobic and hydrophilic contact surfaces and their affinities to viral particles should be investigated. Also, characteristics such as the roughness of ceramic and glass surfaces and the influence they may have had on the retention of proteinaceous soils should be investigated as well.
152
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CONCLUSION
This study demonstrated that QAC and sodium hypochlorite sanitizers normally used to inactivate bacteria (≥ 5 logs) on food contact surfaces in manual and mechanical ware-washing operations are unable to inactivate non-enveloped viruses (≤ 3 logs) at
FDA recommended conditions. Additionally, this study confirmed that the bacteria (E. coli and L. innocua) were more sensitive than the viruses (MNV-1 and PoSaV) to the sanitizing solutions. These findings could be used to improve the current ware-washing protocols in an attempt to help control foodborne infections associated with bacterial and viral contamination at restaurants and other foodservice settings.
Atomic force microscopy (AFM) can be used to evaluate different sanitizing solutions and their ability to remove residual food soils on selected food contact surfaces.
This technique can be used to study the effect that different microorganisms can have on the adhesion of food soils to common food contact surfaces (e.g. stainless steel, glass and ceramics). Also, AFM can potentially be used to determine the relationship between specific sanitizing solutions and contaminating microorganisms to influence the adhesion of selected foods to contact surfaces, thus affecting their removal during ware-washing procedures.
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