EFECTS OF COL WATER WASHING OF SHEL EGS ON HAUGH UNIT, VITELINE MEMBRANE STRENGTH, AEROBIC BACTERIA, YEAST, AND MOLD Except where reference is made to the work of others, the work described in this thesis is my own or was done in collaboration with my advisory commite. This thesis does not include proprietary or clasified information. ______________________________________ Amber Brooke Caudil Certificate of Approval: ___________________________ ___________________________ Omar A. Oyarzabal Patricia A. Curtis, Chair Asistant Profesor Profesor Poultry Science Poultry Science ___________________________ ___________________________ Kenneth E. Anderson Deana R. Jones Profesor Research Food Technologist North Carolina State University Russel Research Center Raleigh, NC Athens, GA ___________________________ George T. Flowers Interim Dean Graduate School EFECTS OF COL WATER WASHING OF SHEL EGS ON HAUGH UNIT, VITELINE MEMBRANE STRENGTH, AEROBIC BACTERIA, YEAST, AND MOLD Amber Brooke Caudil A Thesis Submited to the Graduate Faculty of Auburn University in Partial Fulfilment of the Requirements for the Degre of Master of Science Auburn, AL December 17, 2007 iii EFECTS OF COL WATER WASHING OF SHEL EGS ON HAUGH UNIT, VITELINE MEMBRANE STRENGTH, AEROBIC BACTERIA, YEAST, AND MOLD Amber Brooke Caudil Permision is granted to Auburn University to make copies of this thesis at its discretion, upon request of individuals or institutions and at their expense. The author reserves al publication rights. ________________________ Signature of Author ________________________ Date of Graduation iv VITA Amber ?Brooke? Caudil was born in Wilkesboro, North Carolina on August 21, 1980. She graduated with honors from West Wilkes High School in Milers Crek, North Carolina in May of 1998. On December 18, 2002, she received her Bachelor of Science degre in Poultry Science with a minor in Microbiology from North Carolina State University. She began her graduate studies in the area of egg quality and safety under the direction of Dr. Pat Curtis in the Poultry Science Department at Auburn University in August, 2003. v THESIS ABSTRACT EFECTS OF COL WATER WASHING OF SHEL EGS ON HAUGH UNIT, VITELINE MEMBRANE STRENGTH, AEROBIC BACTERIA, YEAST, AND MOLD Amber Brooke Caudil Master of Science, December 17, 2007 (M.S., Auburn University, 2007) (B. S., North Carolina State University, 2002) 137 Typed Pages Directed by Patricia A. Curtis Most retail shel eggs in the United States are washed in water that can be upwards of 49?C which increases the internal temperature of shel eggs. After procesing, internal egg temperatures may be 6.1 to 7.8?C higher than initial internal egg temperatures. The internal post-procesing temperature of shel eggs fal within the growth range of Salmonela Enteritidis (SE), the most common human pathogen asociated with eggs and egg products. It can take several days for the internal temperature of procesed packaged eggs to reach a temperature that is cool enough to inhibit the growth of most microorganisms, including SE. Washing eggs with cool water vi may be a way to prevent the increase in internal egg temperature during procesing. Experiments were conducted to study the efects of cool water washing on shel egg quality. The presence of aerobic bacteria, yeasts, and molds on exterior shel surfaces, in the contents, and within the shel matrix of eggs were also examined. Egg quality was evaluated by Haugh unit and viteline membrane strength determination. This study was conducted in two phases. Phase one consisted of a pilot study, in which six diferent dual tank wash water temperature combinations, including a single warm water temperature (49?C) and two cool water temperatures (15.5?C and 24?C), were used to wash eggs. The pilot study was conducted in order to identify the best temperature, or combination of temperatures, for washing shel eggs while limiting the increase in the internal egg temperature. Phase two consisted of a commercial study in which shel eggs were washed using four diferent dual tank wash water temperature combinations in two commercial egg procesing facilities. The commercial study examined how commercialy washing shel eggs in cool water afects interior egg quality, as wel as the presence of aerobic bacteria, yeasts, and molds on and within the egg. The pilot study and the commercial study each included ten weks of storage in which the presence of aerobic bacteria, yeasts, and molds on exterior shel surfaces, in the contents, and within the shel matrix of procesed eggs were monitored wekly. Microbial quality was monitored by the USDA griculture Research Service Egg Safety and Quality Research Unit. Egg quality was also monitored during both the commercial and pilot study. During the pilot storage study, no significant diferences in Haugh unit values or viteline membrane strength were found betwen wash water temperature combinations, vii indicating that cool water washing does not afect the egg quality measurements monitored. However, results from the pilot study showed significant diferences (P ? 0.05) in viteline membrane strength and the Haugh unit values as storage time progresed. The average force required to break the viteline membrane decreased 13.9% and average Haugh unit values decreased from 59.2 to 56.4 due to storage. The results of the commercial study indicate that wash water temperature did not significantly afect Haugh unit values or viteline membrane strength. As storage time progresed, however, average Haugh unit values declined 14.8% and the average force required to rupture the viteline membrane decreased 20.6%. Although no significant diferences were found among wash water temperature schemes in amounts of aerobic bacteria, yeast, and mold present on exterior shel surfaces, within the shel matrix, and in egg contents, average amounts of bacteria present on shel surfaces also decreased 11.3% during storage, and bacteria present in egg contents increased 39.5% due to storage. Results of the commercial study indicate that there is a potential for utilizing cool water washing in the commercial seting while stil producing safe eggs. vii ACKNOWLEDGENTS The author would like to thank Dr. Pat Curtis, Dr. Ken Anderson, Dr. Omar Oyarzabal, Dr. Deana Jones, and Dr. Mike Musgrove for their asistance throughout this study. Their patience, support, and guidance have been greatly appreciated. For help in conducting statistical analysis of these data, the author would like to thank Lesli Kerth and Dr. Daryl Kuhlers. The author would also like to thank Dr. Kevin Kener for his engineering asistance, and James Elison, Vanesa Kretzschmar, Alexis Davis, Natasha Sanderfer, and Jesica Moulton for their asistance with laboratory work. Last but not least, the author would like to thank her friends and family for their support and understanding during this endeavor. The author especialy appreciates Jeremy Damron for his asistance and encouragement during the hectic periods of this study. ix Style manual or publication used: Poultry Science Computer software used: Microsoft Word? x TABLE OF CONTENTS LIST OF TABLES.........................................................................................................xi LIST OF FIGURES.......................................................................................................xii I. INTRODUCTION................................................................................................1 I LITERATURE REVIEW......................................................................................5 Formation and Design of the Hen?s Egg................................................................5 Microbial Defenses of the Egg..............................................................................9 Egg Quality........................................................................................................12 Bacteria on & in the Egg.....................................................................................15 Washing & Storing Shel Eggs ............................................................................23 Regulations..........................................................................................................41 II. EFECTS OF COL WATER WASHING OF SHEL EGS ON VITELINE MEMBRANE STRENGTH AND HAUGH UNIT........................46 IV. EFECTS OF COMERCIAL COL WATER WASHING OF SHEL EGS ON HAUGH UNIT, VITELINE MEMBRANE STRENGTH, AEROBIC BACTERIA, YEAST, AND OLD..........................62 V. SUMARY AND CONCLUSIONS.................................................................90 BIBLIOGRAPHY.........................................................................................................98 APENDIX A.............................................................................................................107 APENDIX B.............................................................................................................110 xi LIST OF TABLES II. 1. Wash water temperature combinations used to wash eggs ...........................59 2. Average efects of wash water temperature combination on viteline membrane strength and Haugh unit values..................................................60 IV. 1. Average Haugh unit values, albumen height, and force required to rupture the viteline membrane of eggs from combined procesing facilities for each wek of the storage...............................................................................84 2. Average amounts of yeast present within the shel matrix (interior) and in the contents of procesed eggs for each wek of storage..........................................87 B. 1. Average efects of wash water temperature scheme on Haugh unit values and viteline membrane strength for each procesing facility.............................110 2. Average efects of wash water temperature scheme on albumen height for each procesing facility.......................................................................................111 3. Average efects of wash water temperature scheme on amounts of yeast (log CFU/ml) present on exterior shel surfaces, within the shel matrix, and in contents of eggs procesed at each facility ..................................................119 4. Average efects of wash water temperature scheme on amounts of mold (log CFU/ml) present on exterior shel surfaces, within the shel matrix, and in contents of eggs procesed at each facility...................................................123 5. Average efects of storage time on amounts of mold (log CFU/ml) present on exterior shel surfaces, within the shel matrix, and in contents of eggs procesed at each facility.............................................................................124 xii LIST OF FIGURES II. 1. Parts of the egg................................................................................................6 II. 1. Fabricated pilot egg washer...........................................................................58 2. Average force required to break the viteline membrane of procesed eggs during each wek of storage .........................................................................61 IV. 1. Data logger being placed into a case of procesed eggs.................................81 2. Average post-procesing cooling curves for eggs procesed at Facility A (2a) and Facility B (2b)........................................................................................82 3. Efects of procesing environment on average Haugh Unit values over ten weks of storage (3a) and average amounts of aerobic bacteria present on exterior shel surfaces amongst wash water temperature schemes (3b)..........83 4. Efects of wash water temperature scheme and post-procesing storage time on average amounts of aerobic bacteria present on exterior shel surfaces..........85 5. Efects of wash water temperature scheme and post-procesing storage time on average amounts of aerobic bacteria present on in egg contents.....................86 6. Efects of wash water temperature scheme and post-procesing storage time on average amounts of mold present on exterior shel surfaces (6a) and within the shel matrix (6b) of procesed eggs...............................................................88 7. Efects of wash water temperature scheme and post-procesing storage time on average amounts of yeast present on exterior shel surfaces...........................89 A. 1. Average efects of wash water temperature combination on egg weight over 60 days of storage.......................................................................................107 2. Average egg weight for days 0, 30, and 60.................................................108 3. Average efects of wash water temperature combinations on albumen height over 60 days of storage...............................................................................109 B. 1. Average efects of storage time on albumen height for each facility............112 xii 2. Average efects of storage time on force required to rupture the viteline membrane of eggs from each facility...........................................................113 3. Average efects of wash water temperature scheme on amounts of aerobic bacteria present within the shel matrix (interior) of eggs from each procesing facility........................................................................................................114 4. Average efects of wash water temperature scheme on amounts of aerobic bacteria present in the contents of eggs from each procesing facility..........115 5. Average efects of wash water temperature scheme on amounts of aerobic bacteria present within the shel matrix (interior) of eggs from each procesing facility........................................................................................................116 6. Average efects of wash water temperature scheme on amounts of aerobic bacteria present in the contents of eggs from each procesing facility..........117 7. Average efects of wash water temperature scheme on amounts of aerobic bacteria present on the exterior shel surface of eggs from each procesing facility........................................................................................................118 8. Average efects of wash water temperature scheme on amounts of yeast present on the exterior surface of eggs from each procesing facility...........120 9. Average efects of wash water temperature scheme on amounts of yeast present within the shel matrix of eggs from each procesing facility...........121 10. Average efects of wash water temperature scheme on amounts of yeast present in the contents of eggs from each procesing facility.....................122 1 I. INTRODUCTION Washing shel eggs is somewhat of a controversial subject. The United States, Japan, Australia, and Canada wash shel eggs; whereas, most European countries choose not to wash shel eggs. Some scientists believe that washing shel eggs increase their microbial load. Brooks (1960) concluded that washing shel eggs caused higher bacterial counts when he discovered that the contents of roughly 90% of newly laid eggs were fre from microorganisms and posses natural defenses against bacterial penetration. His discovery helped support the argument for not washing shel eggs, which was based upon the fact that in the absence of water, bacteria are les likely to move through the shel or along the pores (Board et al., 1979). Another negative aspect of washing shel eggs is that the proces can damage the cuticle, which is the egg?s outermost covering and a natural defense against bacterial penetration (Romanoff and Romanoff, 1949; Wesley and Beane, 1967, Sauter et al., 1978; Wang and Slavik, 1998; Favier et al., 2000). Results of many early egg washing experiments indicated that washing increased spoilage, especialy during storage (Lorenz and Star, 1952; Star et al., 1952; March, 1969). Scientists continued, however, to study the efects of diferent washing methods in hopes of reducing, or even preventing, rotting of eggs during storage (Moats, 1979; Lucore et al., 1997; Jones et al., 2004b; Musgrove et al., 2005). The argument for washing shel eggs is based upon the fact that microorganisms from fecal mater, blood, dirt, insects, etc. are found on the shels of eggs. The shels are porous and can be penetrated by 2 bacteria from the shel?s exterior. Also, the nutrients that make eggs a high quality food for humans are also a good growth medium for most bacteria capable of penetrating the shel. Cleaning the shel surface removes potential contamination and reduces the incidence of bacterial penetration, in addition to providing a visualy appealing product for consumers (Moats, 1980; Lucore et al., 1997; Jones et al., 2004b; Musgrove et al., 2005). The federal authority to regulate egg safety is currently shared by the Department of Health and Human Services? Food and Drug Administration (FDA) and the United States Department of Agriculture?s (USDA) Food Safety and Inspection Service (FSIS). The FDA has jurisdiction over the safety of most foods, including shel eggs. The USDA, however, is primarily responsible for implementing the Egg Products Inspection Act (EPIA). This responsibility is shared by FSIS and Agriculture Marketing Service (AMS). The FSIS is responsible for the inspection of procesed egg products in order to prevent the distribution of adulterated or misbranded egg products (USDA, 2003). The AMS conducts a voluntary surveilance program which ensures that participating egg procesors met the USDA?s requirements for plant sanitation, procesing, labeling, refrigeration, and packaging. When eggs are packed under this surveilance program, a USDA grademark can be printed on the carton and the eggs are refered to as ?shielded?. Currently, procesors who chose to produce USDA shielded eggs must abide by specific regulations when washing shel eggs, and are constantly monitored by an AMS inspector while shielded eggs are being produced. Even though the US egg industry washes al table eggs sold to consumers, food safety concerns asociated with the consumption of shel eggs exist. Salmonela 3 Enteritidis (SE) is the most common human pathogen asociated with shel eggs and egg products. SE is one of more than 2,400 strains of Salmonela that can cause an infection known as salmonelosis (Bel and Kyriakides, 2002). Salmonelosis is a bacterial infection that afects the intestinal tract, and occasionaly the bloodstream. Symptoms include severe diarhea, occasional bloody diarhea, fever, chils, abdominal cramps, and vomiting. SE is resilient and able to adapt to extremes in environmental conditions. It can grow within a pH range of 4.5 to 9.5, and in temperatures as high as 54?C (Bel and Kyriakides, 2002). The microorganism, however, does not grow wel at refrigerated temperatures (Gast and Holt, 2000; Rhorer, 1991; Bel and Kyriakides, 2002; Chen et al., 2002). It has been determined that one in 20,000 eggs produced in the United States is internaly contaminated with SE (USDA, 1998). If not safely handled and properly cooked, an egg that is internaly contaminated with SE may result in foodborne ilnes. In the year 2000, an estimated 182,060 ilneses occurred due to egg-asociated SE (Shroeder et al., 2005). Because of the risk of foodborne ilnes asociated with the consumption of shel eggs, the government has made it a top priority to make eggs safe. Emergence of Grade A eggs as a source of SE in the 1980s and 1990s contributed to an increased awarenes of egg safety. This emergence was mainly due to improper handling and preparation of eggs internaly contaminated with SE (St. Louis et al., 1988); however, egg procesing regulations such as the re-washing of eggs and high wash water temperatures were also to blame (Anderson et al., 1992; Meckes et al., 2003). More recent egg washing research (Lucore et al., 1997) has suggested that current egg procesing regulations need to be re-evaluated. 4 Washing, grading, and packaging increases internal egg temperature. Anderson et al. (1992) found that post-procesing internal egg temperatures can be 6.1 to 7.8?C higher than initial internal egg temperatures. Due to current regulations, eggs are washed in water that can be as hot as 49?C. Most shel egg procesors now use dual wash tank systems rather than the single wash tank systems previously used. The dual wash tank system doubles the time that eggs are exposed to hot water spray, which adds to the increase in internal egg temperature (Curtis, 1999). The internal temperature of an egg can continue to rise for up to six hours after the eggs have been placed in a cooler. In fact, it may actualy take the centermost egg in a palet five to six days to reach an internal temperature of 7.2?C (Anderson et al., 1992; Jones et al., 2002b; Chen et al., 2002). This means that for five to six days after procesing, eggs may have an internal temperature that fals within the growth range of SE and other microorganisms. Therefore, failure to cool eggs clearly contributes to the potential for multiplication of SE and other microorganisms if they are present. Washing eggs in cool water may be one way to reduce this problem. 5 I. LITERATURE REVIEW Formation and Design of the Hen?s Egg Since the domestication of the fowl, eggs have been an important part of the human diet. They contribute a number of nutrients to the American diet. Hen eggs contain approximately seventy-five percent water, twelve percent protein, ten percent lipids, and a smal percentage of vitamins and minerals (Gebhardt and Thomas, 2002). They are a nutrient dense source of many esential amino acids, vitamins and minerals. Eggs contain al esential vitamins except vitamin C, and they are one of the few natural sources of vitamins D and B12. Because it is a nutritionaly complete protein containing al of the esential amino acids, egg protein is one of the highest quality proteins available (McNamara, 2004). Based on a diet of 2,000 kcal per day, one large egg provides eleven percent of daily protein needs (Gebhardt and Thomas, 2002). The egg is complex, with many diferent parts. Those parts include the yolk, albumen, shel membranes, shel, and the cuticle (Figure 1). It takes approximately twenty-six hours for a hen to lay one egg. Each part of the egg is formed in a separate section of the hen?s reproductive tract, which is made up of the ovary and the oviduct. In the ovary, ova mature, by acumulating yolk thereby, growing in size. Typicaly, the largest most mature ovum breaks away from a stem connecting it to the ovary and enters the oviduct. The oviduct is the tube through which the egg pases, and where the 6 structures necesary to complete the egg are applied. The oviduct secretes and consecutively applies, in succesion, the albumen, two shel membranes, and the shel. Structuraly, the oviduct is divided into five sections, each having a fairly specific physiological function in the formation of the egg. The oviduct consists of the infundibulum, magnum, isthmus, shel gland (uterus) and vagina. After ovulation takes place, the yolk or ovum is picked up by the infundibulum. The egg then moves from the infundibulum into the magnum, where albumen is secreted and collects in layers around the ovum. The albumen-layered ovum oves from the magnum to the isthmus by peristaltic movement. Addition of two shel membranes occurs in the isthmus. Then, after pasing through the isthmus, the egg enters the uterus where it spends the most time. 7 This time in the uterus alows for adequate calcium deposition to form the shel. Once the shel is complete, the cuticle, which is a thin protective film of transparent material, is applied to the shel?s surface while the egg is in the lower portion of the oviduct (Romanoff and Romanoff, 1949). When the shel is complete the egg moves from the uterus, leaving the oviduct through the vagina, and is expeled through the cloaca. The yolk, which is the center of a freshly laid egg (Figure 1), makes up thirty-one percent of the egg (USDA, 2000). Major components of the yolk are proteins and lipids; nearly al the lipids, vitamins, and minerals found in eggs are located in the yolk. The yolk material is contained in a thin membrane known as the viteline membrane (Figure 1). It is a clear membrane which gives the yolk its shape, and is composed mostly of protein matrix similar to that found in the shel membranes (USDA, 2000). The viteline membrane is made up of thre layers; the outer and inner layers are mucinous and the center layer is composed of keratin (Romanoff and Romanoff, 1949). Its strength prevents the yolk from breaking. Surrounding the yolk is the albumen, which makes up fifty-eight percent of the egg (USDA, 2000). Albumen is made up of approximately forty diferent kinds of proteins, al responsible for its many functional and antimicrobial characteristics. Ovalbumin, ovotransferin, avidin, lysozyme, conalbumin, and ovomucoid are just a few of the proteins found in the albumen. Eggs contain four layers of albumen: an inner thin layer, a thick layer, an outer thin layer (Figure 1), and the chelazaferous (inner thick), which imediately surrounds the yolk and from which the chalazae are created. Located betwen the outer thin layer of the albumen and the internal surface of the shel are the inner and outer shel membranes. The two membranes adhere to each 8 other, and help support the weight of the shel. They also form a complex matrix which deters bacterial penetration. After an egg is laid, the contents cool from the body temperature of the hen to the ambient temperature. As the contents cool, the inner membrane contracts, causing the egg to lose gases and moisture. As this occurs, the two shel membranes separate at the large end of the egg. The outer membrane sticks to the shel and the inner membrane sticks to the egg contents, forming the air cel (Figure 1). The air cel supplies air to the developing embryo when pulmonary respiration is initiated (Romanoff and Romanoff, 1949). The egg shel consists of the inner and outer shel membranes (Figure 1) followed by calcium deposits and diferent shel layers. The eggshel is about ninety-eight percent calcium carbonate in the form of calcite. It also contains magnesium, phosphate, and citrate in smal amounts, as wel as traces of sodium and potasium (Parkhurst and Mountney, 1988). It is 241-371 ?m thick and is perforated with anywhere from 7,000 to 17,000 pores (Tyler, 1961). The thousands of pores are intended to alow for the exchange of respiratory gases, such as carbon dioxide, for the developing embryo (Romanonff and Romanoff, 1949; Wang and Slavik, 1998). The pores also permit the escape of moisture and carbon dioxide from the egg. The outer surface of the shel is covered by a thin (20 to 30 ?m), hard outer protective covering known as the cuticle (Wang and Slavik, 1998). The cuticle (Figure 1) is a thin stratum of minute glycoprotein spheres, and extends a short distance into the pores of the egg (Romanoff and Romanoff, 1949), creating a seal. Imediately after being laid, the cuticle is moist and sticky, but 9 dries and hardens with exposure to air (Romanoff and Romanoff, 1949). The cuticle alows gaseous water to difuse frely through the shel while inhibiting the movement of liquid water into the egg (Sparks and Board, 1984). Microbial Defenses of the Egg The egg is resistant to microbial contamination due to the mechanical and chemical bariers. Therefore, if bacteria are not introduced into the egg during formation, bacterial contamination can only occur after microorganisms encounter overcome these highly eficient bariers. The cuticle, shel, inner and outer shel membranes are the mechanical bariers, and the albumen contains the chemical bariers which are al parts of the egg?s antimicrobial defense system. The cuticle is the egg?s first defense against microbial entry. The cuticle covers the shel and acts as a covering to inhibit bacterial penetration by closing a large portion of the pores within the shel, thereby decreasing shel permeability (Board et al., 1979). However, the cuticle can become damaged as soon as contact is made with the floor of the batery cages, by cleaning methods, harsh detergents, abrasion from washer brushes, and exposure to large amounts of water (Romanoff and Romanoff, 1949; Wesley and Beane, 1967; Sauter et al., 1978; Wang and Slavik, 1998; Favier et al., 2000). A damaged cuticle provides a way for spoilage and pathogenic bacteria to enter the egg (Board, 1966; Wang and Slavik, 1998). The inner and outer shel membranes are two of the most efective bariers to bacterial penetration. They compose the organic matrix of the shel, a glycoprotein fine fibrous net beginning in the basal caps and the inner parts of the mamilae. They are semi permeable and permit pasage of water and crystaloids (Parkhurst and Mountney, 10 1988). Together, the membranes function like a micron filter so extensive that it is uncertain exactly how bacteria manage to penetrate them (Haines and Moran, 1940; Romanoff and Romanoff, 1949; Board and Fuller, 1994; Anderson et al., 2004). The outer membrane is thicker and more porous than the inner membrane, minimizing it?s efectivenes as a barier to bacterial entry. The inner membrane is made up of many protein fibers that are more tightly interwoven; however, it can only delay bacterial entry for a short period of time (Board and Fuler, 1994). This ensures that there are no pores that transverse straight through to the albumen (Wang and Slavik, 1998). Antimicrobial properties of albumen also provide a barier against microorganisms that may have penetrated the mechanical bariers at the egg?s surface (Fleischman et al., 2003). The proteins present in albumen inhibit the growth of a wide variety of microorganisms; whereas, the yolk, or even a mixture of yolk and albumen, are not as efective. Conalbumin is an example of a protein found in albumen that has important antimicrobial properties. The protein chelates metal ions, making them unavailable to bacteria for proliferation. Two other proteins in the albumen with antimicrobial properties are avidin and lysozyme. Avidin can bind to and inactivate biotin, and the lytic action of lysozyme destroys bacteria by causing the cel wal to rupture and disintegrate (Brooks and Taylor, 1955). Lysozyme plays a major role in the defense against Gram-positive bacteria (Board et al., 1986). The change in albumen pH following lay is another barier against bacteria (Haines, 1939; Brooks and Taylor, 1955; Brooks, 1960). After an egg is laid, carbon dioxide moves out of the egg and into the surrounding environment until it?s concentration in the egg and the environment reach equilibrium (Romanof and Romanoff, 1949). The loss of carbon dioxide causes the 11 albumen to become more alkaline. In a newly laid egg, albumen pH is approximately 7.6 (Romanoff and Romanoff, 1949); however, the pH can increase from a fairly neutral pH to a basic 9.7 (Healy and Peter, 1925; Romanoff and Romanoff, 1949). Few bacteria are able to thrive in such a basic environment (Board, 1966). Albumen viscosity is also a barier against bacteria. In fresh eggs, the high viscosity of the albumen and the chalazae anchor the yolk protectively in the center of the egg and hinder movement of microorganisms, especialy motile bacteria, toward the yolk (Board et al., 1986). However, as the egg ages and the albumen becomes more alkaline, the ovomucin- lysozyme complex, or thick gel structure, begins to break down and the albumen becomes les viscous (Romanoff and Romanoff, 1949; Board, 1966; Wiliams, 1992). This reduced viscosity makes it easier for microorganisms to spread inside the egg (Chen et al., 2005). The viteline membrane, which keeps the yolk confined and separate from the albumen (Board and Fuller, 1974), is also one of the egg?s many defenses against microbial contamination. The viteline membrane prevents the sepage of yolk into the albumen, and is responsible for preventing the entry of bacteria into the yolk. Because the nutrients present in yolk make it a good growth medium for bacteria that may be present in the egg?s albumen, the viteline membrane plays an important role in the egg?s microbial integrity. If the membrane breaks, or even stretches enough to alow yolk into the albumen or bacteria into the yolk, the yolk wil provide nutrition to any bacteria present (Conner et al., 2002). 12 Egg Quality Egg quality is based on the characteristics of an egg that afect its aceptability to the consumer (Watkins, 2004). Prior to the emergence of Grade A shel eggs as a potential source of SE contamination, consumers defined egg quality in physical and visual terms, and few consumers expresed concern about the microbial load contained on or within commercialy procesed eggs. Today, internal egg quality is defined as a function of physical, functional, and microbiological quality. External egg quality is a function shel structure, physical quality, and microbiological quality. Physical quality refers to shel characteristics such as soundnes, shape, thicknes, texture, and cleanlines. Functional quality refers to characteristics such as albumen viscosity, yolk color, viteline membrane strength, and how wel an egg performs in a food system. Microbiological quality refers to the absence of pathogenic bacteria. After an egg has been laid, the rate of deterioration wil never fully stop, and can only be slowed or delayed (Anderson et al., 2004). Internal egg quality decline occurs when the thick gel structures of the albumen thin and become watery, causing water to migrate to the yolk. Osmotic movement of water across the viteline membrane leads to a flatened and enlarged yolk, as wel as a stretched and consequently weakened viteline membrane (Romanoff and Romanoff, 1949). The changes in the quality of the albumen and yolk are a function of temperature, reduced carbon dioxide, increased pH, egg age, and the loss of moisture (Romanoff and Romanoff, 1949; Wiliams, 1992; Chen et al., 2005; Samli et al., 2005). Determining the Haugh unit value is the most common way to ases interior egg quality. The USDA-AMS has acepted the Haugh unit as a valid and reliable method for 13 determining interior egg quality. The Haugh unit is used to determine albumen quality; and it is considered the standard for shel egg interior quality measurement. Haugh (1937) discovered that the change in quality or condition of an egg varies as a negative logarithm and not as a linear function. In order to establish an acurate index of egg quality in which the numerical value would equal the quality value, he developed the Haugh unit (Haugh, 1937). The Haugh unit is a relationship betwen egg weight and the height of the thick albumen. There are, however, limitations asociated with the Haugh unit measurements. Scientists have argued that the calculation used to determine the Haugh unit is inacurate for eggs other than size large (Silversides et al., 1993). This is due to the fact that the calculation is weighted exclusively for a 56.7g (2oz) egg (size large). The questioned validity of the Haugh unit as an acurate indicator of interior egg quality is why Silversides et al. (1993) suggested only measuring albumen height as a means of determining egg quality. A year later, Siversides and Vileneuve (1994) reported that albumen height and the Haugh unit value equaly describe albumen quality. Recent studies, however, have found that measuring the height of the inner thick albumen introduces a bias against old hens and some hen strains (Silversides and Scott, 2001). The egg?s internal temperature when the Haugh unit value is being determined can also negatively afect the Haugh unit value in terms of being an acurate indicator of quality (Kener et al., 2006). In order to acurately and consistently determine Haugh unit values, eggs should be cooled to an internal temperature betwen 7.2 and 15.6?C, and the internal temperature of those eggs must be uniform (USDA, 2000). Another common indicator of internal egg quality is the strength of the viteline membrane. Viteline membrane strength is commonly measured using static 14 compresion (Conner et al., 2002; Jones et al., 2002b; Kener et al., 2006). A machine applies presure to the yolk at a specified rate until the viteline membrane is ruptured. The amount of presure/force required to rupture the yolk corresponds to the viteline membrane strength. The more force required, the stronger the viteline membrane (Jones et al., 2002b). As the egg ages, the albumen pH increases due to the loss of carbon dioxide and water moves from the albumen into the yolk; the viteline membrane is eventualy afected by the alkaline pH and becomes weak (Romanoff and Romanoff, 1949; Wiliams, 1992; Chen et al., 2005). As previously mentioned, additional water increases the size and weight of the yolk, which in turn stretches the viteline membrane. The yolk appears flatened and the membrane can easily break (Romanoff and Romanoff; 1949). Conner et al. (2002) found that after eight weks of storage in an environment with an ambient temperature of 10?C, the force required to rupture the viteline membrane declined from 2.33 to 1.56 grams. A weak viteline membrane can be viewed as an indicator of potential microbial contamination, as wel as poor physical, quality (Gast and Beard, 1990; Humphrey et al., 1991; Humphrey, 1994; Chen et al., 2005). The disintegration or weakening of the viteline membrane as the egg ages makes it possible for microorganisms to invade the egg yolk (Chen et al., 2005). If the viteline membrane breaks, or even stretches enough to alow sepage of the yolk into the albumen, the yolk not only provides nutrition to any bacteria present (Conner et al., 2002); it also afects the egg?s functional properties. Albumen that has been contaminated by even the smalest amount of yolk, for example, loses some of its whipping/foaming characteristics due to the lipid content of the yolk (Romanoff and Romanoff, 1949). 15 Bacteria on and in the Egg Despite the egg?s many microbial bariers, bacteria are stil able to penetrate the shel and membranes. Factors that improve bacteria survivability on the shel surface, reducing the egg?s antimicrobial defense system, include the physical condition of the cuticle and underlying shel (Sparks and Board, 1984); the presence of water on the shel (Board et al., 1979); and the concentration of iron in water that comes into contact with the egg (Board et al., 1986). If the cuticle is damaged or washed away, the pores are exposed, and there is a greater susceptibility to microbial entry into the contents (Board, 1966; Wang and Slavik, 1998). The diameters of pores range from 9-35 ?m (Romanoff and Romanoff, 1949), which is significantly larger than most microorganisms (which are typicaly 1-5 ?m). Salmonela species, for example, range from 0.7-1.5 ?m wide and 2.0- 5.0 ?m long (Bel and Kyriakides, 2002). Because pores are larger in size, Salmonela species and other bacteria found on the shel can move through them into the contents and cause spoilage. Microorganisms found on egg shels are capable of breaching the shel?s microbial bariers. These microorganisms are mainly Gram-positive bacteria derived from dust, soil and feces (Haines, 1939; Zasgaevsky and Lutikova, 1944; Board, 1964, 1966). The dominant contaminants on the shel tend to be Gram-positive cocci and bacilus such as Micrococcus and Arthrobacter (Hutchinson et al., 2003). Once the shel?s microbial bariers have been breached, Gram-negative bacteria are more capable of withstanding the antimicrobials present in the albumen (Board, 1966; Jones et al., 2004a); therefore, the internal contaminants of eggs are commonly Gram-negative organisms such as Alcaligenes, Achromobacter, Pseudomonas fluorescens, Salmonela, 16 and Eschericia (Hutchinson et al., 2003). A study conducted by Jones et al. (2002a) found that SE and Psudomonas fluorescens were both able to survive at diferent rates in various parts of the egg. While SE survived best on the exterior surface of the shel, Pseudomonas fluorescens was beter able to transverse the shel membranes and infect the contents of the egg. Florian and Trussel (1957) identified Pseudomonas fluorescens as a primary invader of the inner shel membranes and predicted that its presence alows other organisms, refered to as secondary invaders, to breach the membranes. These secondary invaders are only able to pas thru the membranes once mechanical bariers, such as inner shel membrane, have been breached by primary invaders (Florian and Trussel, 1957). Over the years, eggs have changed in a number of ways. They have become larger and rounder in shape (Curtis, 1999; Anderson et al., 2004). Tharington et al. (1999) noted that genetic improvements in commercial layer strains have impacted egg size. The study suggested that in the past forty years eggs have become larger and contain a smaler percentage of yolk, which in turn, results in a lower percentage of yolk fat. These genetic improvements have made eggs more susceptible to microbial penetration (Curtis, 1999). Jones et al. (2002a) found that for some historic layer strains, a decrease in the microbial integrity of the eggs may have acompanied the genetic changes at these points in time. They suggested that screning for microbial integrity should be included in the selection proces among laying hen breders. The results of a study conducted by Jones et al. (2004a) indicate that genetic selection over time has altered eggs? ability to withstand microbial contamination and penetration during storage. 17 The authors suggest that factors such as porosity of the shel, thicknes of the shell membranes, and concentration of natural antimicrobials may have been altered by genetic selections. Although the egg industry washes al table eggs sold to consumers, potential food safety concerns asociated with the consumption of shel eggs exist. An estimated one in 20,000 eggs in the United States contain SE, and can cause ilnes if eaten raw or not thoroughly cooked in foods before consumption (USDA, 1998). Each year, Salmonela species are implicated in approximately 50,000 cases of bacterial food poisoning in the United States (Meckes et al., 2003). Salmonela bacteria have been known to cause ilnes for over one hundred years. SE is the most common human pathogen asociated with shel eggs and egg products. SE is a Gram-negative, motile, rod-shaped bacterium. It can grow under aerobic and anaerobic conditions, is resilient, and able to adapt to extreme environmental conditions. The microorganism can survive and grow at temperatures as high as 54?C. SE growth in eggs, however, is inhibited at temperatures of 7.2?C and below (Rhorer, 1991; Curtis, 1999; Bel and Kyriakides, 2002; Chen et al., 2002). SE can be transmited from the laying hen to the egg either as a result of fecal contamination or infection of the oviduct. If SE is present on the egg?s shel, there is the potential for the contents to become infected as wel. Gast and Beard (1990) reported a correlation betwen egg shels contaminated with SE and SE positive feces from artificialy infected hens. Eggs can also be infected with SE during formation. This can occur if the intestinal tract of a hen is colonized with SE. The SE, if present, can then migrate into the reproductive tract, where possible contamination of yolk, albumen, or both can occur (Gast and Beard, 18 1990). If the ovary of a hen is infected with SE, during egg formation, the yolk (ova) may become seded with SE cels before leaving the ovary or while pasing through the oviduct. When this occurs, the egg typicaly contains low numbers of SE cels when it is laid (Humphrey et al., 1989, 1991; Gast and Beard, 1992; Chen et al., 2002). If eggs or egg products containing live Salmonela bacteria in high enough populations are consumed, an ilnes known as salmonelosis can occur. Salmonelosis is one of the more common foodborne ilneses in the US. Foods asociated with salmonelosis are those of animal origin, fruits, and vegetables have al been found at some point to be contaminated with Salmonela. Some foods of animal origin commonly asociated with salmonelosis include poultry, milk and dairy products, eggs, and seafood (Bel and Kyriakides, 2002; CDC, 2003a; USDA, 2005). Symptoms of the ilnes usualy develop within 8-72 hours after ingesting the bacteria. Diarhea, fever, abdominal cramps, chils, headache, nausea, and vomiting are al symptoms of salmonelosis; they typicaly last four to seven days (Bel and Kyriakides, 2002; CDC, 2003a; USDA, 2005). A total of 5,198 laboratory-diagnosed cases of foodborne Salmonela infections occurred during 2001 (CDC, 2002). Because mild cases are typicaly not diagnosed or reported, the actual number of infections may be thirty or more times greater (CDC, 2003a). Approximately twenty percent of the population is considered to be at a higher risk for salmonelosis because they are imuno-compromised (USDA, 1998). Imuno- compromised individuals include the very young, the very old, hospital patients, nursing home residents, and individuals with compromised imune systems. Salmonela infections can be life-threatening for the imuno-compromised. It is estimated that approximately six hundred imuno-compromised individuals die each year with acute 19 salmonelosis (CDC, 2003a). Most people recover from salmonelosis without any long- term health problems; however, about two percent of those who do recover may later develop recurring joint pains and arthritis. The annual cost asociated with human salmonelosis due to SE is estimated to range from $150 milion to $870 milion (USDA, 1998). From 1976 to 1986, reported SE infections increased more than six fold in the northeastern United States. From January, 1985 to May, 1987 65 foodborne outbreaks of SE, asociated with 2119 cases and eleven deaths, were reported. Seventy-seven percent of the outbreaks with identified food vehicles were caused by Grade A shel eggs or foods that contained such eggs (St. Louis et al., 1988). In 1999, there were nineten outbreaks of salmonelosis in the United States. Of those nineten outbreaks for which a vehicle could be confirmed, fiften (79%) were asociated with shel eggs (Meckes et al., 2003). In 2001, state and local health departments reported 46 confirmed outbreaks of SE infection to CDC. A food vehicle was confirmed for 24 of the 46 outbreaks. Eggs were an ingredient in 15 (63%) of the 24 confirmed vehicles (CDC, 2003b). Due to the increasing number of human ilneses asociated with the consumption of SE contaminated shel eggs, in December of 1996 the FSIS and the FDA joined together in order to develop a comprehensive risk asesment of SE. The goals of the SE Risk Asesment included determining the total risk of foodborne ilnes caused by SE, identifying and evaluating possible strategies to reduce the risk of SE contamination, identifying areas in which future research was needed, and prioritizing future data collection eforts (USDA, 1998). In order to best determine the total risk of SE related foodborne ilnes, the Risk Asesment consisted of five modules. Those modules were: 20 (1) Egg Production Module, (2) Shel Egg Module, (3) Egg Products Module, (4) Preparation and Consumption Module, and (5) Public Health Module. The Egg Production Module estimated the number of eggs produced that were internaly contaminated or infected after lay with SE. The Module estimated that on average 3.3 milion SE positive eggs are produced from the 65 bilion eggs laid in those years (USDA, 1998). The Shel Egg Module, the Egg Products Module, and the Preparation and Consumption Module estimated the increase or decrease in the number of SE organisms present in eggs or egg products as they pased through storage, transportation, procesing, and preparations. The Shel Egg Module followed shel eggs from collection through procesing, transportation, and storage. Important components of this model were the amount of time required for loss of the viteline membrane?s integrity and the growth rate of SE in eggs after the viteline membrane?s breakdown. The Egg Products Module tracked the change in the amount of SE present in further egg procesing facilities from receiving thru pasteurization. The Preparation and Consumption Module explained that extended storage times and ambient temperatures encouraged the growth of microorganisms that might be present in the contents of eggs. When identifying and evaluating possible strategies to reduce the risk of SE contamination, the Shel Egg and Egg Products Modules determined that the use of multiple interventions/precautions would result in a more substantial reduction in SE ilneses than simply using one intervention/precaution by itself. Two interventions which showed the most potential for reducing the number of SE ilneses asociated with the consumption of contaminated 21 eggs were: (1) lowering the temperature in which shel eggs were maintained, and (2) diverting eggs produced by SE-positive flocks from the shel egg market to the pasteurized, egg products market (USDA, 1998). The Public Health Module calculated the frequency of SE ilneses, as wel as the clinical and possible long-term outcomes of those ilneses. In addition to the SE Risk Asesment, President Clinton established a Council on Food Safety in August, 1998. The Councils? main goals were to reduce and prevent the incidence of human salmonelosis and to protect the health of American people by preventing foodborne ilnes using wel-coordinated surveilance, investigation, inspection, enforcement, research, educational programs, and science-based regulation. Preventing human salmonelosis includes benefits such as reducing economic losses asociated with the reduction of productivity linked to human ilnes, reducing pain and suffering, and reducing expenditures on medical treatment (USDA, 1998). In order to identify gaps in the scientific community?s understanding of SE and its route of on-farm transmision, the President?s Council of Food Safety created the Egg Safety Action Plan (USDA, 1998). In August of 1999, the President?s Council on Food Safety held a public meting in order to obtain input during development of the Egg Safety Action Plan. Representatives from consumer groups and the egg industry came to the conclusion that the federal government needed a set of mandatory national standards which would asure consumers that al eggs across the United States were subject to the same safety standards. In order to help met their goals, the Council of Food Safety commisioned the Egg Safety Task Force. The Egg Safety Task Force included federal food safety agencies responsible for egg safety. FDA, CDC, FSIS, APHIS, AMS, and ARS, were responsible 22 for developing an action plan to eliminate eg-asociated SE ilneses. The Egg Safety Action Plan included a farm-to-table continuum which focused on preventing SE contamination of eggs on the farm prior to lay, after eggs have been laid, during procesing, and following procesing, as wel as promoting the use of safe egg handling practices by food preparers in the retail industry and in homes across America. The overal public health goal of the Egg Safety Action Plan is to eliminate SE ilneses asociated with the consumption of eggs by 2010. When developing the Egg Safety Action Plan, one responsible agency for each stage of the farm-to-table continuum was identified based on the strengths of each agency. The FDA?s responsibilities included developing standards for the producer, and enforcing those standards by requiring States to provide on-farm inspections. The FSIS was responsible for developing standards for both shel egg packers and egg products procesors. The FDA and CDC were responsible for conducting surveilance and monitoring activities. The Egg Safety Action Plan gave the egg industry a choice betwen two SE reductions strategies. Those strategies included a SE testing-egg diversion system on the farm, or a lethal treatment or ?kil step? at the packer/procesor. Both strategies required regulatory personnel to be present on the farm and at the packer/procesor. In 2005, two new risk asesments, SE in shel eggs and Salmonela species in egg products, were created with information obtained after the release of the 1998 SE Risk Asesment (USDA, 2005). These new risk asesments predicted that pasteurization and rapid cooling of eggs would be the most efective means of reducing ilneses from SE contaminated eggs and egg products contaminated by Salmonela species. The SE in shel eggs asesment estimated that storing and holding eggs at 23 7.2?C within 12 hours of lay would reduce human ilneses from 130,000 to 28,000 per year (USDA, 2005). The Salmonela species in egg products asesment concluded that the annual number of human ilneses would be reduced from 130,000 to 41,000 if al eggs produced in the US were pasteurized for a 3-log 10 reduction of SE (USDA, 2005). The risk asesments also identified several opportunities for further research. These opportunities include a nationaly representative survey for the prevalence of SE in domesticaly produced flocks, hens, and shel eggs; a characterization of growth parameters of SE in shel eggs; a quantitative study of cross-contamination during shel egg and liquid egg product procesing; studies on how SE difers from other Salmonelae in ability to persist in chicken reproductive tisue and egg contents; and a characterization of egg storage times and temperatures on farms and in homes, for eggs produced off-line and for eggs at retail (USDA, 2005). Washing and Storing Shel Eggs As previously mentioned, washing eggs has not always been an acepted means of cleaning and preserving them; however, washing shel eggs has been a common practice in America since the mid-20 th century. Before modern egg procesing technology, farmers momentarily dipped their eggs in boiling water in order to preserve them (Board, 1966). The late 1800?s and early 1900?s marked the beginning of modern egg production. The first mechanical continuous egg washing systems were developed in the 1950s (Hutchinson et al., 2003). At that time, the most common type of egg washer was a wire basket that could hold 50-60 eggs at one time. The basket was manualy lowered into a rotating washing machine. A household dish or laundry detergent was added, and the eggs were submerged and agitated for approximately one to thre minutes 24 before being removed (Hutchinson et al., 2003). This type of washing is refered to as static or imersion washing. In 1959, methods for rapidly washing large quantities of eggs using imersion washing for mas procesing were developed (Lucore, 1994). A study conducted by Lorenz and Star (1952) compared bacterial loads of spray washed eggs and imersion washed eggs. They found that spray washing drasticaly reduced the percentage of spoiled eggs during storage. This is because the cuticle wil cease to exist if it is wet for an extended period of time (Board, 1966, 1979; Wang and Slavik, 1998). It was also discovered that static water in washing machines produced more spoilage than sprayed water (Lorenz and Star, 1952). This is due to a negative presure gradient that is created when eggs are fully submerged in water that is slightly cooler than that of the eggs. The negative presure causes wash water, as wel as any bacteria present in that water, to be pulled into the eggs. In 1975, static, or imersion, washers were banned and replaced by spray washers (USDA, 1975). Not only were imersion washers banned, but it also became ilegal to soak eggs as a means of cleaning them. However, it is currently not ilegal for a farmer to clean eggs by imersing them in water before the eggs are sent to the procesing plant. There have been significant changes over the past forty-plus years in egg procesing. One such change has been the industry?s shift from of-line production, where eggs are placed on flats or carts at the farm and transported to the procesing facility two to thre times a wek, to in-line production, where multiple houses in a single complex are connected by a common egg belt which transports eggs directly from the layer house into the procesing facility. In-line production has enabled the egg industry to get eggs from the bird to the consumers? table in a shorter period of time (Curtis, 25 1999). In the past 15 years, other changes in egg procesing have included the use of computer controlled, high speed, high volume egg washers (Knape et al., 2002). Washing shel eggs is not only a way to reduce the risk of pathogenic bacteria from being on the egg shel, it also provides a clean, visualy appealing product for consumers. In the United States, as in most countries, customers demand eggs that are visibly clean, making it dificult to sel dirty eggs. Therefore, despite potential pitfals, a number of countries such as the United States, Canada, Sweden, Australia, and Japan have embraced egg washing. Although Star et al. (1952), Lorenz and Star (1952), and March (1969) found that washed eggs suffered more bacterial spoilage than unwashed eggs, Forsythe et al. (1953) reported that washing can efectively remove over 80 to 90% of shel contaminates when using diferent types of chemical agents. The opposing results were due to the washing method. Forsythe et al. (1953) utilized a method that involved lightly brushing eggs while a stream of water flowed onto them; whereas, the others washed eggs by imersion washing. Moats (1979) also showed that washing under commercial conditions (which at that time included spray washing rather than imersion washing) was highly efective in reducing surface bacterial counts on egg shels to low levels. In fact, washing has been shown to reduce the number of microorganisms on egg shels from 43,000 per shel to les than 10 (Lucore et al., 1997). Musgrove et al. (2005) found that current commercial practices decrease the prevalence of eggs contaminated with aerobic bacteria by thirty percent. More importantly, current commercial practices have been found to reduce the number of aerobes present on eggs by 99.9% (Musgrove et al., 2005). In countries where egg washing has become a routine 26 and established practice, it is regarded as safe, and is perceived by consumers as an esential part of the hygienic production of eggs (Hutchinson et al., 2004). In the United States, most shel eggs are washed using a general proces that involves four stages: weting, washing, rinsing, and drying. Most commercial facilities spray wash eggs using a dual wash tank system. Typicaly, the eggs are placed on rollers which act as a conveyor belt. The rollers cary the eggs through the four stages of the washing proces. The first stage of egg washing is weting, or pre-washing, which softens any debris that may be on the egg shel. The eggs then go through two diferent wash tanks. In each wash tank the eggs pas under rotary or reciprocating brushes while they are sprayed with warm wash water. The rollers that the eggs are caried on continuously turn the eggs, enabling al surfaces of each egg to be exposed to the brushes and warm water spray (Hutchinson et al., 2003). The wash water with which the eggs are sprayed with is continuously re-circulated. A food grade detergent is added to the wash water in order to help remove fecal mater, blood, dirt, stains, etc., and to maintain a high pH (>10.5). It is important to maintain a high pH in egg wash water in order to maintain low counts of total aerobic bacteria in wash water (Bartlet et al., 1993). Once the eggs have been washed, they are rinsed with high presure jets of warm water and sanitizer. Rinsing the eggs removes any loose debris that may have been picked up by the eggs during the main washing proces. Rinsing also removes any residues left by detergents and defoamers in the wash water, and helps decrease the risk of cross contamination asociated with the brushes used during the main washing proces (Hutchinson et al., 2003). After being rinsed, the eggs are blown dry. Once the eggs have been blown dry, they are graded, packed, and placed in a cooler or shipped directly to a retail outlet. 27 Acording to Bel et al. (2001) and Paterson et al. (2001), shel eggs are purchased by the consumer within an average of nineten days after they have been procesed. The major parameters influencing egg washing are: wash water quality and mineral content, wash chemicals, pH of wash water, and temperature of the wash water (Hutchinson et al., 2003). The hardnes of the water entering the procesing facility can have a dramatic impact on the ability of detergents and sanitizers to operate properly (Jones et al., 2003); therefore, it is important that wash water not be too hard. Natural or artificial contamination of wash water with iron salts results in a high incidence and fast rate of egg spoilage. Research conducted by Garibaldi and Bayne (1960, 1962) asociated the presence of iron in egg wash water with increased spoilage in washed eggs. When iron is present in the wash water, it reverses the bacteriostatic action of an antimicrobial known as conalbumin, which is found in the egg?s albumen (Garibaldi and Bayne, 1962). Iron, which is an esential trace nutrient, is required by many microorganisms in order to grow. Once iron is introduced into the egg, one of the egg?s microbial defenses, the bacteriostatic action of conalbumin, is useles and microorganisms are able to grow due to the availability of an esential trace nutrient (Garibaldi and Bayne, 1960, 1962). Current USDA regulation (7 CFR 56.76(e)(6) requires shel egg procesors producing USDA shielded eggs to conduct an analysis of the iron content of their water supply. If the iron content exceds two parts per milion, the regulation requires the provision of equipment to correct the exces iron content. Defoamers also play an important role in egg washing. Defoamers are chemicals that are added to egg wash water because one of the main functional properties of eggs is as a foaming agent in food preparation. During the washing proces, eggs can be broken; 28 therefore, re-circulated wash water typicaly contains albumen. Egg foam is created when air is incorporated into the proteins and water of egg albumen. The washing proces incorporates air into the re-circulated wash water which contains albumen, and foam can be created. Without the proper addition of defoamers to the wash water, foam wil build up in the wash tanks and eventualy overflow. When foam spils from the tanks, it can interfere with the level, pH, and temperature of the wash water. Detergents are wash chemicals which are added to the wash water in order to elevate the pH. They are dispensed, for the most part, in concentrations necesary to clean the egg shel (Curtis et al., 2004). Most procesing facilities continuously monitor the amount of detergent present, and have machines that automaticaly dispense detergent when needed. Moats (1978) found that eggs washed in water containing a sanitizing chemical invariably spoiled les that eggs washed in water alone. Wash water pH is also an important egg washing parameter. Catalano and Knabel (1994) reported that maintaining wash water conditions at pH 11 or above prevents possible cross- contamination caused by recycled wash water by efectively reducing the number of SE present on egg shels and in wash water. When studying various combinations of wash water temperature and pH, Kinner and Moats (1981) found that at a pH ranging from 10 to 11 the amount of bacteria present in wash water decreased, regardles of water temperature (35, 40, 45, 50, or 55?C). Although the temperature of wash water is an important egg washing parameter, if it is more than 4.5 to 10?C above the temperature of the eggs being washed, thermal cracks may occur. Thermal cracks occur when the egg contents expand and actualy cause the shel to crack. Decreasing the bacterial load of procesed eggs is more eficiently acomplished by controlling pH, rather than increasing 29 wash water temperature. It has been shown, however, that when no or improper control over wash water pH and temperature is used, the eggs can have a higher bacterial load after being washed than before (March, 1969; Moats, 1978). This is most likely due to being washed with re-circulated wash water containing a high bacterial load. Controlling wash water pH is also a means of controlling bacterial growth in re-circulated wash water. The egg shel is sensitive to acid, and may become damaged or disolve if it is exposed to a relatively strong acid for any extended amount of time. Because of eggs? sensitivity to acid, the pH is controlled using alkaline detergents. When used acording to manufacturers? recommendations, alkaline detergents produce an initial pH in the wash water near 11, and help to maintain the pH in the 10-11 range during washing. Raising the pH of wash water to 10-11 significantly reduces the number of organisms, such as coliforms, present in the wash water and has been shown to kil Salmonela species which could potentialy contaminate clean egg shels (Kinner and Moats, 1981; Catalano and Knabel, 1994). Pearson et al. (1987) reported that egg wash water of high pH was bacteriostatic to E. coli and Salmonela, and suggested that HACP programs involve regular sampling and analyses. Barlet et al. (1993) also reported that there is a strong relationship betwen a pH equal to or greater than 10.5 and low counts of total aerobic bacteria in wash water sampled from commercial facilities. Holley and Proulx (1986) found that when the wash water pH was 9.5 or les, Salmonela species were able to survive in wash water with a temperature as low as 42?C. Because the detergent plays such an important role in egg washing, it is equaly important to use the right type of detergent. Quaternary amonium compounds, 30 chlorine, sodium carbonate, sodium hydroxide, sodium hypochlorite, and potasium hydroxide are some examples of commonly used egg washing detergents. Unfortunately, detergents which may be efective in reducing the bacterial load found on eggs may also damage the egg?s cuticle or shel (Sauter et al., 1978; Wang and Slavik, 1998; Favier et al., 2000; Hutchinson et al., 2004). In order to study the efects of chemicals used in egg washing on microstructural changes of eggshels, Wang and Slavik (1998) washed eggs using thre common commercial egg washing detergents - a quaternary amonium compound, sodium carbonate, and sodium hypochlorite. Their washing proces was conducted in a laboratory seting and took 3.5 minutes. The washing time included 2.5 minutes for brushing and rinsing and one minute for blow-drying. They found that while the quaternary amonium compound and sodium hypochlorite cleaned the eggs without causing excesive damage to eggshel surfaces, sodium carbonate, removed large parts of the eggshel surface layer and most of the cuticle layer. Wang and Slavik (1998) concluded that diferent degres of cuticle damage can be produced on eggshel surfaces by diferent types of egg washing chemicals, and that altered eggshel surfaces may alow greater microbial penetration. Despite the possible pitfals asociated with alkaline detergents, if the right type of detergent wil physicaly remove or inactivate up to 92% of the bacteria on an eggshel?s surface without damaging the cuticle (Forsythe et al., 1953; Bierer et al., 1961; Wang and Slavik, 1998). Detergents used to wash eggs should be food safe and compatible with the eggs, the washing equipment, and any other chemicals used in the washing proces (Hutchinson et al., 2003). Despite how wel an egg is washed, storage wil cause a decline in egg quality and slowly breaks down the egg?s natural bariers, making it increasingly susceptible to 31 bacterial entry and growth (Brooks and Taylor, 1955; Board, 1966; Kim et al., 1989; Humphrey, 1994; Wang and Slavik, 1998; Jones et al., 2004b). As early as the mid 1900?s, scientists (Lorenz and Star, 1952; March, 1969) observed changes that occurred in washed eggs during storage. These changes caused increased bacterial infections, and eventualy lead to spoilage. In 1989, Kim et al. reported that various characteristics of albumen and yolk quality are lost as eggs age. When an egg is newly laid, the yolk is located in a central position. The central position of the yolk is primarily due to the support it receives from the albumen, and is regarded as an indicator of high quality. During storage, however, the albumen begins to break down and is no longer able to provide as much support for the yolk. This results in the increased movement of the yolk, which indicates poorer quality (Board, 1966). Jones et al. (2002b), Jones and Musgrove (2005), and Samli et al. (2005) have al reported a decrease in Haugh unit values during storage due to the break down of albumen. Also, when an egg is newly laid, the viteline membrane is strong and prevents the yolk from seping into the albumen. However, Eliot and Brant (1957), Hartung and Stadleman (1963), Jones et al. (2002b), and Chen et al. (2005) have al found that storage length negatively afects viteline membrane strength. The changes that occur to an egg?s internal components during storage not only result in a decline in quality; they also cause the egg to become more susceptible to bacterial growth. Humphrey et al. (1991) reported that egg age can impact SE growth. Humphrey (1994) and Jones et al. (2004a) reported that SE contamination of egg contents increased during storage at 20?C and 26?C, respectively. When studying bacterial penetration into washed eggs stored at diferent temperatures and times, Wang and Slavik 32 (1998) found that storage time was an important factor for Salmonela penetration into egg contents; the longer the storage time, the more the Salmonella penetration. The high temperatures and high pH of egg wash water kil most, but not al of the microorganisms present on egg shels. The microorganisms that are not kiled are physiologicaly damaged. An extended storage period gives these injured microorganisms time to rejuvenate. Once rejuvenated, they are beter able to work their way through the shel membranes and into the albumen. Another factor that increases the susceptibility of eggs to bacterial growth during storage is the breakdown of the albumen. As previously mentioned the albumen in fresh eggs is highly viscous and anchors the yolk in the center of the egg, thus hindering the movement of microorganisms toward the yolk (Board et al., 1986). Not long after an egg is laid, chemical changes cause the gel structure of the albumen to break down, and the albumen becomes les viscous (Romanoff and Romanoff, 1949; Board, 1966; Wiliams, 1992). The relatively high pH of albumen creates an unfavorable growth environment for most microorganisms; however, when albumen viscosity changes, motile bacteria that may be present are les restricted and able to migrate into eggs? contents more easily (Chen et al., 2005). As previously discussed, the viteline membrane becomes weak and also begins to break down during storage. Scientific studies have shown that egg age has an obvious impact on the ability of SE to grow rapidly in albumen adjacent to the yolk (Humphrey, 1994). Conner et al. (2002) found that the ability of SE to grow in albumen coresponds to a decline in the force required to break the viteline membrane. An aged and weakened viteline membrane becomes permeable and may alow bacteria to enter the yolk, yolk contents to enter the albumen, or both (Humphrey, 1994; Conner et al., 2002; 33 Chen et al., 2005). Studies using eggs from artificialy (Gast and Beard, 1990) and naturaly SE infected (Humphrey, 1994) hens have shown the albumen next to the viteline membrane to be an important SE contamination site. Scientists have also found that SE wil grow wel near the viteline membrane, but wil not grow in areas away from the membrane (Murase et al., 2005). Kim et al. (1989) reported that Salmonela are severely inhibited and sometimes kiled by conalbumen or ovatransferin found in high concentrations in the albumen. The ovatransferin chelates iron and generaly prevents bacterial growth; however ovatransferin does not prevent growth of bacteria on the yolk surface (Kim et al., 1989). Researchers have found that egg yolk supports rapid microbial growth (Clay and Board, 1991; Humphrey and Whitehead, 1993; Gast and Holt, 2000), and that the multiplication of microorganisms located in albumen does not occur until the bacteria present have acesed the yolk (Sharp and Whitaker, 1927; Gast and Holt, 2000). This is because egg yolk is rich in iron and contains nutrients needed to support the rapid growth of bacteria (Clay and Board, 1991; Humphrey and Whitehead, 1993; Gast and Holt, 2000). As yolk components migrate into albumen, bacteria that have previously exhausted the albumen?s iron reserve have a renewed supply (Schaible et al., 1944; Humphrey, 1994). Although SE cels require iron to grow, they generaly cannot make use of iron present in the yolk of fresh eggs because the viteline membrane prevents the entry of bacteria into yolk contents as wel as the release of iron into the albumen (Humphrey, 1994). If, however, contact with yolk contents does occur and permisive temperatures exist, the egg becomes an environment in which SE can grow rapidly (Conner et al., 2002). A recent study conducted by Gast et al. (2005) found that 34 SE and Salmonela Heidelberg deposited outside the viteline membrane of freshly laid eggs is sometimes able to reach yolk contents and begin to multiply within a day of storage at a warm temperature. Because high wash water temperatures currently required by USDA regulations increase internal egg temperature (Anderson et al., 1992), they can acelerate the rate of functional decline and microbial growth (Wiliams, 1992; Lucore et al., 1997; Fleischman et al., 2003). As the temperature of egg wash water rises, there is an increased risk of cuticle damage and thermal cracking (Wesley and Beane, 1967). Cuticle damage and thermal cracking provide ways for spoilage and pathogenic bacteria, especialy from the egg wash water, to enter the egg. High wash water temperatures also cause the internal temperature of eggs to rise. In 1955, Hilerman reported that wash water maintained at 46.1?C would increase internal egg temperatures by 0.22?C per second. Anderson et al. (1992) found that post-procesing internal egg temperatures can be 6.1 to 7.8?C higher than initial internal egg temperatures. In addition to the initial rise due to procesing, an egg?s internal temperature can continue to rise for up to six hours after being placed in a cooler (Anderson et al., 1992). In a more recent study, Jones et al. (2002b) found that after procesing, shel eggs required at least five days to reach an internal 7.2?C when stored at 7.2?C. This means that for five or more days after procesing, eggs may have an internal temperature that fals within the growth range of SE and other microorganisms (Anderson et al., 1992; Chen et al., 2002; Jones et al., 2002b). After being procesed, eggs are typicaly packaged in cartons or flats, 30 dozen eggs (in cartons or flats) are placed into cases, and then 30 cases are paletized. These packaging 35 conditions help to ensure that the increase in internal egg temperature wil be maintained for several days. Feddes et al. (1993) found that eggs packed in cases cool at a rate that is seven times slower than uncased eggs. Czarick and Savage (1992) suggested that the use of solid cardboard cases be abandoned if the goal of the egg industry is to obtain egg temperatures of 7?C as rapidly as possible. It is also possible that the heat from high wash water temperatures not only increases the internal temperature of the egg, but weakens the viteline membrane as wel (Fleischman et al., 2003). Research conducted by Kinner and Moats (1981), Holley and Proulx (1986), and Lucore et al. (1997) suggests that wash water temperatures commonly used by most egg procesors is neither hot enough to kil microorganisms on the shel nor cool enough to inhibit their growth. High egg wash water temperatures serve to increase internal egg temperatures, and act as an added buffer to prevent rapid cooling of the egg; thus alowing organisms on the shel, as wel as inside the egg, to continue to grow (Lucore et al., 1997). The dual wash tank system, commonly used by most egg procesors, forces the eggs stay in a hot, wet environment for a longer period of time (Curtis, 1999), which adds to the increase in internal egg temperature. SE contaminated eggs typicaly contain les than one hundred cels per egg at the time of lay (Humphrey, 1994); however, if an egg is contaminated with SE, increased internal egg temperature caused by high wash water temperatures, combined with the break down of the egg?s antimicrobial defenses, provide SE cels opportunity to rapidly grow. The rate of the viteline membrane deterioration, for example, is increased when the egg is exposed to high storage temperatures. Proper refrigeration has been shown to slow quality decline (Conner et al., 2002; Chen et al., 2005). Eggs should be stored in a cool environment in 36 order to reduce loss of moisture, reduce albumen thinning, slow weakening of the viteline membrane, and most importantly, to prevent/reduce microbial growth (Conner et al., 2002; Chen et al., 2005). Researchers have discovered that if SE is present in egg contents, the bacteria?s growth rate directly responds to the temperature at which the eggs were stored. In 1989, Kim et al. found that as storage temperatures increased, the growth rate of SE in eggs did as wel. They concluded that storage temperature is the most important factor in determining the growth response of SE in eggs. Gast and Holt (2000) reported dificulty in promoting SE growth in eggs stored betwen 10 and 17.5?C. Other scientists have found that storage temperatures of 7.2?C and below reduce the colonization and subsequent growth of Salmonela in eggs (Rhorer, 1991; Bel and Kyriakides, 2002; Chen et al., 2002). Humphrey et al. (1989) reported that storing eggs at refrigerated temperatures causes SE to be more susceptible to the high temperatures used in cooking eggs. The Shel Egg Procesing and Distribution Module within the SE Risk Asesment found an eight percent reduction in foodborne ilneses when eggs are maintained at an ambient temperature of 7?C throughout shel egg procesing and distribution (USDA, 1998). Storing eggs at 7?C or below combined with quickly reducing the internal egg temperature, also serves to prevent the growth of any bacteria that may be lodged in pores of the egg shel. In addition to making the egg an inhospitable environment for most bacteria, reducing post-procesing internal egg temperature as quickly as possible could also have a positive impact on egg quality. As previously discussed, viteline membrane strength and albumen quality are both influenced by internal egg temperature. 37 The importance of internal egg temperature has led scientists to developed methods to quickly reduce eggs? post-procesing internal temperature (Curtis et al., 1995; Thompson et al., 2000). Thompson et al. (2000) found that a properly managed forced- air system could quickly cool packaged eggs. Curtis et al. (1995) discovered that cryogenic gases could quickly cool eggs before packing. In 2002, Jones et al. (2002b) reported that egg quality was enhanced by quick cooling and exposure to gaseous carbon dioxide. Unfortunately, each of the methods developed require the use of additional equipment and changes in plant design. The extra costs asociated with these methods have detered egg procesors from using them. Gast et al. (2006) reported that the efectivenes of refrigeration for limiting bacterial multiplication in eggs is dependant upon initial level and location of contamination, movement of bacteria or nutrients within the egg, and the rate at which growth-restricting temperatures are achieved. Although procesors have litle control over initial level and location of contamination as wel as movement of bacteria and nutrients within the egg, they can more easily control the amount of time needed to achieve growth-restricting temperatures. However, current shel egg procesing regulations, combined with the current technology, limits the procesors? ability to lower the internal egg temperature in a very short period of time (Curtis, 1999). As previously mention, shel eggs are generaly purchased by the consumer within an average of nineten days after being procesed (Bel et al., 2001; Paterson et al., 2001). Because most eggs reach the retail outlet in such a short period of time, reducing their internal temperature to 7.2?C or below can be chalenging. It has been suggested that washing eggs in cool water, as opposed to warm water, could aid in reaching and maintaining 38 growth-inhibiting internal egg temperatures of 7?C or below. Current regulations, however, require egg wash water to be 90?F (32.2?C), or 20?F (11.1?C) warmer than the warmest egg, and maintained at that temperature (7 CFR 56.76(f)(3). Research supporting this regulation was conducted in the mid 1900?s. In 1940, Haines and Moran reported that egg wash water colder than internal egg temperature causes the negative presure gradient previously discussed. Research conducted by Lorenz and Star (1952) concluded that eggs washed in cold water were more likely to spoil than eggs washed in warm water. In 1948, Funk presented data which indicated that when the temperature of the wash water was lower than the internal temperature of the egg, losses in storage were definitely greater compared with storage losses in eggs washed in water warmer than internal egg temperature. However, a similar group of experiments conducted at a diferent time found that storage losses among washed dirty eggs were not influenced by the temperature of the wash water (Miler, 1954). The specifics of the wash water temperature regulation, however, are mainly based on research conducted by Brant and Star (1962) and Brant et al. (1966). Their research concluded that the temperature of the wash water should be greater than 11?C warmer than the egg temperature. There is, however, a problem with research supporting the current wash water temperature regulation. When the research was conducted, the most common way to wash eggs was by imersion washing. Eggs were completely submerged in water and agitated for one to thre minutes. As previously mentioned, eggs are currently spray washed and never fully imersed in wash water. Recent research conducted on the efects of lower wash water temperatures is rather contradicting. In 1997, Lucore et al. presented good evidence that cooler wash 39 water temperatures do not contaminate shel eggs in any greater amount than warm to hot temperatures. They also recommended a re-examination of cold water washing procedures. Using pilot egg procesing equipment and a spray wash system in a pilot plant, Lucore et al. (1997) compared the efects of thre wash water temperatures upon internal and external shel surface bacterial counts. They reported that internal microbial counts from eggs spray washed with water as cool as 15.5?C were no diferent from internal microbial counts of eggs spray washed with 48.9?C water. In a more recent inoculation study (conducted in a laboratory seting), Hutchinson et al. (2004) reported that wash and rinse water temperatures did not significantly efect surface populations of SE. They also, however, reported that alowing wash and rinse water temperatures to fal below 34?C caused a detectable amount of content contamination. Although it is not clear why, it is possible that the results reported by Lucore et al. (1997), contradict the findings of Hutchinson et al. (2004) due to a diference in wash water pH, a diference in washing environment and equipment (pilot egg procesing equipment in a pilot plant versus a laboratory seting), or because the temperature of only the wash water was lowered and the rinse water temperature remained consistent with USDA guidelines (7 CFR 56.76(f)(11)). Lucore et al. (1997) also found that cool water washing aided in reducing the internal temperature of eggs once they have been washed, packaged, and placed into the cooler. This, in turn, reduces the amount of time needed to cool eggs, and appears to reduce microbial contamination levels by inhibiting their growth (Lucore et al., 1997). There is also a possibility that washing eggs in cool water could help maintain, or even enhance, interior egg quality during storage. More rapid cooling of the 40 egg to refrigerated temperatures may help maintain viteline membrane strength, and possibly decrease the chances of any nutrients becoming available for microbial growth. Previous research conducted to determine the efects of cool water washing of shel eggs has been performed in a laboratory seting and has not taken into acount the bacteria found in recycled wash water utilized in commercial procesing facilities. High wash water temperatures are not only used as a means of preventing the entry of bacteria into eggs, but also as a means of controlling the bacteria found in the re-circulating tank. Research conducted by Kinner and Moats (1981) showed that at a neutral pH, the temperature range used to wash eggs is not lethal to most types of bacteria. They found that rapid bacterial multiplication occurred at pH 7 and 8 at a temperature range of 35 to 45?C; however, at a pH of 10 and 11 bacterial numbers decreased at al temperatures used in the study (35, 40, 45, 50, and 55?C). In 1994, Leclair et al. studied the efects of wash water temperatures ranging from 38?C to 46?C and pH ranging from 9.5 to 10.5 on the inactivation of S. typhimurium and L. monocytogenes. They found that recycled wash water required significant increase in temperature (47.4?C), as wel as pH (10.8), in order to eliminate the two pathogens. That same year, however, after washing artificialy contaminated eggs in 37.7?C water at pH 9 and 11, Catalano and Knabel (1994) found that the higher pH significantly reduced external SE contamination. They reported that high pH prevents possible cross-contamination caused by recycled wash water by efectively reducing the number of SE present on egg shels and in wash water. The research conducted by Kinner and Moats (1981) and Catalano and Knabel (1994) suggest that if pH is controlled, and the wash water temperature lowered, it is possible to get the same bacterial kil level without excesively increasing the internal temperature of the 41 eggs. Previous research has determined that spray washing eggs in cool wash water does not increase internal bacterial counts of shel eggs; however its afect on bacteria found in the re-circulating tank remains unknown. Cool water washing of shel eggs in a commercial seting, rather than a laboratory seting, wil give beter insight into its afect on bacteria found in recycled wash water and commercialy procesed eggs. The intended purpose of cool water washing of shel eggs is to help reduce internal egg temperatures during and after procesing and possibly prevent the multiplication of SE if it is present. Ataining growth-inhibiting temperatures of 7?C or below shortly after procesing wil reduce the probability that consumers wil be exposed to amounts of pathogenic bacteria present in egg contents sufficient to cause foodborne disease. In addition to initiating the egg cooling proces and shortening the cooling time, a cool water wash could benefit egg procesors by reducing, or even eliminating, the cost of heating wash water and by decreasing the amount of energy needed to cool eggs following procesing. Cool water washing could also be economicaly beneficial to the egg industry by reducing wear and tear on refrigeration units in cooler rooms. Regulations The egg industry became large enough to warant regulatory intervention from the government in 1910, when egg consumption exceded 300 eggs per capita (Lucore, 1994). In 1928, the USDA began the inspection of eggs. In the 1950?s the USDA placed requirements on the washing and sanitizing of shielded shel eggs (Lucore, 1994). Further regulations dealing with egg procesing were introduced in 1967. These regulations required that continuous-typed washers have the wash water changed once per shift; however, specifications as to the length of time for a shift were not included 42 (Lucore, 1994). In 1970, the Egg Products Inspection Act (EPIA) was pased (USDA, 2003). The EPIA was designed to prevent the marketing of checks, dirties, leakers, losses and inedible eggs to the consumer. Implementation and enforcement of the EPIA is the primary responsibility of the USDA. The act requires commercial flocks of more than 3,000 hens to be registered with the USDA. Producers that have 3,000 laying hens or more and any egg handler or distributor that sorts and segregates eggs for sale to the consumer are subject to mandatory inspections. These mandatory inspections are conducted at least once per quarter by Federal or State inspectors. The responsibility of implementing and enforcing the EPIA is currently shared by the FSIS and Agriculture Marketing Service (AMS). In order to ensure that only eggs fit for human consumption are used for such purposes, the FSIS conducts mandatory surveilance of egg packers. The AMS conducts a voluntary surveilance program that ensures participating egg procesors met USDA requirements for plant sanitation, procesing, labeling, refrigeration, and packaging (USDA, 2007). When eggs are packed under this surveilance program, a USDA grader must be present and an official USDA grademark can be printed on the carton. These eggs are refered to as ?shielded?. Procesors who chose to produce USDA shielded eggs must abide by specific USDA regulations (USDA, 2007). One regulation pertains to the recycling of wash water. As previously discussed, egg wash water is continuously recycled in order to achieve beter use of limited amounts of water. There is, however, an increase in bacterial numbers in the recycled water due to the fact that the recycled water is warm and caries an organic load. In an atempt to reduce the potential hazards of recycling 43 wash water, the government requires egg procesors to empty their old wash water and replace it with clean water every four hours or more often if needed to maintain sanitary conditions, and at the end of each shift (7 CFR 56.76(f)(5). In addition to removing the organic load caried by recycled wash water, replacing used wash water with clean water (including detergent) helps ensure that the wash water is at a pH of 10 or greater. Most procesing facilities continuously monitor the amount of detergent present, and have machines that automaticaly dispense detergent when needed. Another regulation states that the wash water temperature must be at least be 90?F (32.2?C), or 20?F (11.1?C) warmer than the warmest egg entering the procesing line, and that this temperature must be maintained (7 CFR 56.76(f)(3). The most recent USDA regulation, which applies to al shel eggs, states that eggs must be stored in a post-procesing environment of 7.2?C or cooler (9 CFR 590.50(a). Because SE does not grow wel at refrigerated temperatures (Gast and Holt, 2000; Bel and Kyriakides, 2002), the post-procesing refrigeration temperature requirement serves as a means to control potential foodborne pathogens asociated with eggs. The federal authority to regulate egg safety is currently shared by the USDA and the Department of Health and Human Services? Food and Drug Administration (FDA). The FDA has jurisdiction over the safety of foods in general, which includes shel eggs. With regard to eggs and egg products, the FDA?s top priority is their safety. One way the FDA ensures the safety of eggs and egg products is by enforcing federal labeling requirements (21 CFR 160). They also require retail establishments to refrigerate shel eggs as soon as they are received and continue to store them in an environment with an ambient temperature of 7.2?C or cooler (21 CFR 115.50). These regulations are intended 44 to help reduce the incidence of SE in eggs; thus, making eggs safer for consumers. In order to improve egg safety, the FDA also investigates SE outbreaks that are due to foods in interstate commerce. If eggs have been implicated in any of those SE outbreaks, the FDA is responsible for performing trace backs in order to identify the source of those eggs. In order to prevent foodborne ilnes, it is imperative to lower post-procesing internal egg temperatures as quickly as possible. Current shel egg procesing procedures and regulations, however, are responsible for a significant increase in internal egg temperatures during and after procesing. Packaging materials then act as insulation and make it dificult to rapidly reduce internal egg temperatures. As previously discussed, research conducted in the 1990?s found that spray washing shel eggs in cool water did not increase internal shel bacterial counts. If fact, the cool water aided in reducing internal egg temperatures following procesing and packaging (Lucore et al., 1997). There is also a possibility that washing eggs in cool water could help maintain, or even enhance, interior egg quality during storage. Cool water washing could also provide economic benefits to the egg industry by reducing, or even eliminating, the cost of heating wash water and decreasing the amount of energy needed to cool eggs following procesing. The objectives of the following research are to determine if cool water washing of shel eggs alters levels of microbial populations, enhances egg quality, and provides a positive economic impact for the shel egg industry. The efects of cool water washing of shel eggs have been determined for eggs washed in a laboratory seting; 45 however, the efects are not known for eggs procesed in commercial procesing facilities. Because of this, a large part of the subsequent research occurs in a commercial seting. 46 II. EFECTS OF COL WATER WASHING OF SHEL EGS ON VITELINE MEMBRANE STRENGTH AND HAUGH UNIT VALUES ABSTRACT SE is currently the most common human pathogen asociated with shel eggs and egg products. Its growth is inhibited at temperatures of 7.2?C and below. Because today?s egg washing proces can increase internal egg temperature 6.7 to 7.8?C, obtaining internal egg temperatures of 7.2?C and below can be dificult. Washing eggs at a cooler temperature could speed the reduction in internal egg temperature, and in turn, reduce potential SE growth by preserving interior quality factors such as viteline membrane strength and Haugh unit. A pilot study was conducted to determine if washing eggs in cool water would alow for more rapid cooling of eggs and possibly afect interior egg quality. Six diferent dual tank wash water temperature combinations, which included a single warm water temperature (49?C) and two cool water temperatures (15.5?C and 24?C), were used to wash eggs. A storage study followed, in which the viteline membrane strength was monitored wekly for ten weks, and Haugh unit values were determined for days 0, 30, and 60 post-procesing. Wash water temperature did not significantly afect viteline membrane strength or Haugh unit values. There were, however, significant diferences (P ? 0.05) in the force required to break the viteline membrane and Haugh unit values due to storage. The average force required to break the 47 viteline membrane decreased 13.9% due to storage, and average Haugh unit values decreased from 59.2 to 56.4 by day 60. (Key words: shel eggs, cool wash, egg quality, egg procesing) 48 INTRODUCTION Procesors who chose to produce USDA ?shielded? eggs must abide by specific USDA regulations. One such regulation states that egg wash water must be at least be 90?F (32.2?C), or 20?F (11.1?C) warmer than the warmest egg entering the procesing line (7 CFR 56.76(f)(3). Due to this regulation, eggs from in-line operations (hen houses directly connected to the procesing facility) can be washed in water as hot as 48.9?C. The most recent regulation pertaining to egg procesing applies to al shel eggs and requires eggs to be stored in a post-procesing environment of 7.2?C or cooler (USDA, 1999). Because scientists have found that the growth of Salmonela Enteritidis (SE), the organism ost often asociated with foodborne disease and eggs, is inhibited at temperatures of 7.2?C and below (Rhorer, 1991; Bel and Kyriakides, 2002; Chen et al., 2002), the post-procesing refrigeration temperature requirement serves as a means to control potential foodborne pathogens asociated with eggs. Washing, grading, and packaging, however, can cause post-procesing internal egg temperatures to be 6.1 to 7.8?C higher than initial internal egg temperatures (Anderson et al., 1992). The internal temperature of an egg can continue to rise for up to six hours after procesing, packaging, and being placed in a cooler (Anderson et al., 1992). It can take five or more days for the centermost egg in a palet to reach an ambient temperature of approximately 7.2?C (Anderson et al., 1992, Jones et al., 2002b; Chen et al., 2002); therefore, for five or more days after procesing, eggs may have an internal temperature that fals within the growth range of SE and other microorganisms. Reducing post-procesing internal egg temperatures as quickly as possible wil help prevent and inhibit the growth of any foodborne pathogens that may be present in egg contents. 49 The increase in internal egg temperature during and after procesing can be atributed to the high temperatures currently used in egg washing. In 1981, research conducted by Kinner and Moats found that wash water bacterial counts decreased, regardles of the temperature, when the water was at a pH of 10 and 11. Washing in warm water increases internal egg temperature and serves as an added buffer to prohibit quick cooling of the egg; thus alowing organisms on the shel, as wel as inside the egg, to continue to grow (Lucore et al., 1997). Due to the increasing number of human ilneses asociated with the consumption of SE contaminated shel eggs, scientists have been focusing on finding ways to reduce the egg?s internal temperature during and after procesing. Methods based on the use of cryogenic gases as wel as forced cool air to rapidly cool shel eggs post-procesing have been developed (Curtis et al., 1995 and Thompson et al., 2000, respectively). Although it has been shown that egg quality is maintained or even enhanced by these methods of rapid cooling (Curtis et al., 1995; Thompson, et al., 2000; Jones et al., 2002b), the methods require additional equipment and some alteration of plant design. Due to cost and space constraints, their use by the egg industry has been limited. Because washing in warm water increases internal egg temperature, serves as an added buffer to prohibit quick cooling of the egg, and in turn, alows organisms on the shel and inside the egg to continue to grow (Lucore et al., 1997), research has been conducted to determine the possibility of preventing excesive increases in internal egg temperature during procesing through cool water washing. Lucore et al. (1997) found that spray washing eggs in 15.5?C wash water did not increase the internal bacterial counts of shel eggs. They also reported decreased bacterial counts on egg shels as wash water temperature 50 decreased. Lucore et al., (1997) concluded that cooler wash water temperatures help reduce the amount of time needed to cool eggs, and they recommended a re-examination of cold water washing procedures. Reducing post-procesing internal egg temperature as quickly as possible may also help enhance egg quality. Two common ways to ases the interior quality of an egg are measuring the force required to break the viteline membrane and determining the Haugh unit value (HU). Viteline membrane strength has become increasingly important for food safety reasons (Mesens et al., 2005). The viteline membrane surrounds the yolk and is responsible for separating the yolk from the albumen (Board and Fuller, 1974). Its strength is an important quality factor because it protects the yolk from breaking or leaking nutrients into the albumen and possibly alowing bacteria to penetrate the yolk. Viteline membrane strength is influenced by internal egg temperature (Fleischman et al., 2003) and storage time. As the egg ages, viteline membrane strength declines (Conner et al., 2002; Jones et al., 2002a; Chen et al., 2005). The membrane also breaks down faster at higher storage temperatures (Romanoff and Romanoff, 1949; Chen et al., 2005). The degradation of the viteline membrane can also afect the functional properties of the egg. Albumen that has been contaminated by even the smalest amount of yolk, for example, loses some of its whipping/foaming characteristics due to the lipid content of the yolk (Romanoff and Romanoff, 1949). Determining the HU is a common way to ases interior egg quality (Haugh, 1937) and has been acepted by USDA-AMS as a valid and reliable method (USDA, 2000). The HU value is a function of egg weight and the height of the thick albumen (Haugh, 1937). Although the HU is commonly used to measure interior quality, there 51 are limitations asociated with HU measurements. The calculation used to determine the HU is weighted exclusively for a 56.7g (2oz) egg (size large); which is why Silversides et al. (1993) questioned the validity of the HU as an acurate indicator of interior egg quality. They argued that the calculation was inacurate for eggs other than size large and suggested measuring albumen height in order to determine interior quality. More recently, however, scientists have reported that albumen height and the HU value equaly portray albumen quality (Silversides and Vileneuve, 1994). Like viteline membrane strength, the HU tends to decline as the egg ages (Wiliams, 1992; Jones et al., 2002b; Jones and Musgrove, 2005; Samli et al., 2005). The purpose of this study was to identify the best temperature, or combination of temperatures, for washing shel eggs while limiting the increase in the internal egg temperature. This study also intends to determine if cool water washing of shel eggs in the pilot seting impacts egg quality. MATERIALS AND METHODS Washing Eggs Nest run shel eggs were purchased from a local packer and identified as originating from a single laying flock. Before being washed, al eggs were stored on nest run carts at 7.2?C. Eggs were washed using a fabricated pilot egg washer which was designed to miic commercial wash conditions (Figure 1). The pilot washer was a stainles stel unit with eleven, six wide egg rollers (Sanova Engineering Corp, Elk Grove Vilage, IL). One row of rollers was used for the drive belt and rotated the eggs during the washing proces (26 rpm). Spray nozzles were mounted in the top of the unit, and positioned in a way that ensured each egg was sprayed with wash water. The spray 52 nozles? presure averaged 4 psi. In order to miic commercial wash conditions, the pilot washer was designed as a dual tank washer and the wash water was recycled. One aspect of the pilot washer that did not miic commercial wash conditions was its lack of brushes. For thre consecutive days (replicates), eggs were washed using six wash water temperature combinations (n = 50 eggs/wash). As sen in Table 1, each temperature combination consisted of a temperature for the first and second wash tank. A single warm water temperature of 48.9?C was utilized along with two cool water temperatures of 15.5?C and 24?C. The single warm water temperature of 49?C was utilized because it represents the warmest temperature commonly utilized by shel egg procesing facilities in order to met USDA regulations. The two cool water wash temperatures were selected based on the limitations to cool water in the commercial procesing facility. The pH of the wash water was maintained betwen 10.5 and 11.5 in order to miic commercial wash conditions. Each day, one cart (5400 eggs/cart) of the nest run shel eggs was procesed. Only one third of the eggs were utilized in determining egg quality; the remaining two thirds were split, with one third utilized as untreated controls and for aerobic population determinations and one third inoculated with SE (Jones et al., 2005). During procesing, eggs were exposed to the wash water spray for a total of one minute (30 seconds per wash tank). Imediately after washing, the eggs were sprayed with a 49?C sanitizing solution that contained 200 ppm chlorine, in acordance with USDA guidelines (7 CFR 56.76(f)(11)). After being sprayed with sanitizer, the eggs were asepticaly removed from the rollers, randomly placed into new foam cartons, and alowed to air dry before 53 the cartons were closed. A ten wek storage study followed, in which the cases of eggs were stored on palets at 7.2?C until analysis. During the storage study, the presence of aerobic bacteria and SE, the viteline membrane strength, and HU values were monitored wekly. Wekly aerobic population and SE determination was conducted by the USDA?s Egg Safety and Quality Research Unit. Results from the microbial analysis are reported in a separate manuscript (Jones et al., 2005). Measuring Viteline Membrane Strength Each wek, a 12-egg sample from each temperature combination was removed from storage and candled; al cracked eggs were excluded from testing. Viteline membrane strength was determined using a TA-XT2i texture analyzer (Texture Technologies, Scarsdale, NY). A texture analyzer determines viteline membrane strength using static compresion (Conner et al., 2002; Jones et al., 2002b; Kener et al., 2006). Each egg was individualy broken into a shalow dish and the yolk was positioned under a 1mm, rounded end, stainles stel probe. Because Lyon et al. (1972) reported that the strongest section of the viteline membrane in near the chalazae, care was taken to ensure that measurements were not obtained from this area. Direct presure was applied to the yolk until the viteline membrane ruptured and the probe penetrated the yolk. Compresion measurements were made using a 5 kg load cel (calibrated using a 2 kg weight), 0.1 gram trigger force, and 3.2 m/sec test speed. The viteline membrane breaking strength was recorded as grams of force required to rupture the membrane. The force required to break the viteline membrane corresponds to its strength; a strong membrane requires more force to break. 54 Determining Haugh Unit Values On days 0, 30, and 60 of storage, a 12-egg sample from each temperature combination was removed from storage, candled, cracked eggs were excluded, and the HU value for each egg was recorded. With the asistance of a QCD instrument range (Technical Services and Supplies, Dunnington, York, England), HU values were determined using procedures based on the formula described by Haugh (1937). Statistical Analysis The data collected was analyzed using SAS (1999). HU values and force required to rupture the viteline membrane were analyzed acording to the general linear model. Any means that were found to be significantly diferent (P ? 0.05) were separated using the least-squared means option of the general linear model procedure. RESULTS AND ISCUSION Although eggs washed in temperature combination 5 averaged the greatest force required to rupture the viteline membrane (1.56 g), wash water temperature configuration did not significantly afect viteline membrane strength (Table 2). Ten weks of storage, however, caused a steady decline in viteline membrane strength (Figure 3). As storage time progresed, the average force required to break the viteline membrane decreased 13.9%. Like viteline membrane strength, there were no significant diferences betwen wash water temperature combinations in average HU values (Table 2). Eggs washed in temperature combination 1, however, had the lowest average HU value (54.3) and eggs washed in the temperature combinations 4 and 5 had the highest average HU values (61.3, 61.4 respectively). Although initial HU values were poor and equivalent to USDA 55 Grade B quality (USDA, 2000), there were significant diferences in average HU values as storage time progresed. The average HU value was 59.2 on the day of procesing, but actualy increased to 61.5 after 30 days of storage. After 60 days of storage, the average HU value decreased to 56.4, a 4.7% decline from the initial value and a significant 8.3% decline from the average value after 30 days of storage. The decline in viteline membrane strength and HU values observed in the current study was not surprising. As early as the mid 1900?s, scientists such as Lorenz and Star (1952) and March (1969) had observed that changes occur in washed eggs during storage. As previously mentioned, scientists have found that extended storage causes viteline membrane strength and Haugh unit values to decline (Eliot and Brant, 1957; Hartung and Stadleman; 1963; Wiliams, 1992; Conner et al., 2002; Jones et al., 2002b; Chen et al., 2005; Jones and Musgrove, 2005; Samli et al., 2005). In addition to causing a decline in egg quality, storage slowly breaks down the egg?s natural bariers and causes the egg to become increasingly susceptible to bacterial entry and growth (Board, 1966; Humphrey, 1994). Some scientists suggest that the degradation of the viteline membrane provides nutrients for SE growth (Conner et al., 2002; Fleischman et al., 2003) because a weakened viteline membrane cannot prevent yolk from seping into the albumen. If yolk is introduced into the albumen, the yolk negatively afects many of the albumen?s antimicrobial properties. This is due to the fact that yolk contents are rich in iron, which SE cels require in order to grow, and provide nutrients that serve as a growth medium for Salmonela organisms that previously exhausted the iron reserves of the albumen (Humphrey, 1994). 56 The decline of an egg?s internal quality occurs when the thick gel structures of the albumen become thin and the viteline membrane becomes weak. The albumen pH of a newly laid egg is approximately 7.6 (Romanoff and Romanoff, 1949); however, as the egg ages, the albumen becomes more alkaline and may increase to approximately 9.7 (Healy and Peter, 1925; Romanof and Romanoff, 1949). Few bacteria are able to thrive in such a basic environment (Board, 1966). As the albumen becomes more alkaline, the gel structure begins to break down, causing the thick albumen to thin and become watery (Romanoff and Romanoff, 1949; Wiliams, 1992). When this occurs, water is absorbed from the albumen into the yolk, causing the yolk to increase in size and weight. The yolk?s increased weight and size causes the viteline membrane to stretch and weaken (USDA, 2000). Because the rate of interior egg quality decline increases as the environmental temperature rises (Romanoff and Romanoff, 1949; Kim et al., 1989; Chen et al., 2002), quickly reducing the post-procesing internal egg temperature can help maintain internal egg quality. These data indicate that wash water temperature does not afect average viteline membrane strength and HU values, and suggest that cool water washing has the potential to improve interior egg quality. As sen in Table 2, eggs washed using temperature combination 1, which is commonly utilized by egg procesors, had the lowest average HU values. Also, eggs washed using temperature combinations containing only cool water temperatures (4 and 5) had the greatest average viteline membrane strength and HU values. Cool water washing of shel eggs could alow for more rapid cooling after procesing while maintaining interior egg quality. Maintaining interior egg quality characteristics, especialy the integrity of the viteline membrane, combined with 57 reducing the eggs? internal temperature wil aid in retarding the growth of any potential pathogenic bacteria present. Jones et al., (2005) found that al wash water temperature combinations investigated in this study were equaly capable of removing SE. Data colected during this study suggest that there is a potential for utilizing cool water washing in the commercial seting while stil producing quality eggs that are microbiologicaly safe for consumption. Washing shel eggs in cool water could also be economicaly beneficial to the egg industry by reducing the energy needed to heat wash water and cool eggs after they have been procesed and packaged. A commercial study wil be conducted in order to beter determine the efects of cool water washing on interior egg quality, aerobic bacteria, yeast, and mold presence, and the frequency of Salmonela, Campylobacter, Listeria, and Enterobacteriaceae. ACKNOWLEDGENTS This research project was funded through a cooperative agrement (Agrement number 58-6612-2-215) betwen the USDA gricultural Research Service and the National Aliance of Food Safety and Security. The authors would like to thank Alan Savage and Tim Brown for designing and fabricating the pilot egg washer used for this study. The authors would also like to thank Patsy Mason, Susan Akins, Jordan Shaw, Lesli Kerth, Vanesa Kretzschmar, and Abby Stewart for their technical asistance. 58 Figure 1. Fabricated pilot egg washer 59 Table 1. Wash water temperature combinations used to wash eggs Combination Tank 1 (?C) Tank 2 (?C) 1 49 49 2 49 24 3 49 15.5 4 24 24 5 15.5 15.5 6 24 15.5 60 Table 2. Average efects of wash water temperature combination on viteline membrane strength and Haugh unit values Temperature Combination Viteline Membrane Force (g) Haugh Unit 1 1.54 54.5 2 1.50 59.4 3 1.53 58.8 4 1.50 61.3 5 1.56 61.4 6 1.53 58.7 SEM 0.02 1.86 61 1.30 1.40 1.50 1.60 1.70 1.80 2 3 4 5 6 7 8 9 Storage Time (week) Force (g) Figure 2. Average force required to break the viteline membrane of procesed eggs during each wek of storage* *There is no data for storage weks 0, 1, and 10 due to technical dificulties. 62 IV. THE FECTS OF COMERCIAL COL WATER WASHING OF SHEL EGS ON HAUGH UNIT, VITELINE MEMBRANE STRENGTH, AEROBIC BACTERIA, YEASTS, AND MOLDS ABSTRACT Current egg washing practices utilize wash water temperatures averaging 49?C, and have been found to increase internal egg temperature by 6.7 to 7.8?C. These high temperatures create a more optimal environment for bacterial growth, including Salmonela Enteritidis (SE), if it is present. SE is the most common human pathogen asociated with shel eggs and egg products. Its growth is inhibited at temperatures of 7.2?C and below. This study?s objective was to determine if commercialy washing eggs in cool water would aid in quickly reducing internal egg temperature, preserving interior egg quality, and creating an environment les beneficial to bacteria. During thre consecutive days, eggs were washed using four dual tank wash water temperature schemes (H = 49?C, 49?C; HC = 49?C, 24?C; C = 24?C, 24?C; CH = 24?C, 49?C) at two commercial procesing facilities. A ten wek storage study followed, in which viteline membrane strength, Haugh unit, and presence of yeast, mold, and aerobic bacteria were monitored wekly. As storage time progresed, average Haugh unit values declined 14.8%, the average force required to rupture the viteline membrane decreased 20.6%, average amounts of bacteria present on shel surfaces decreased 11.3%, and bacteria present in egg contents increased 39.5% due to storage. Wash water temperature did not significantly afect Haugh unit values, viteline membrane strength, or the 63 amounts of aerobic bacteria, yeast, and mold within the shel matrix of procesed eggs. Results of this study indicate that incorporating cool water into commercial shel egg procesing, while maintaining a pH of 10 to 12, lowers post-procesing egg temperatures and alows for more rapid cooling, without causing a decline in egg quality or increasing the presence of yeast, mold, and aerobic bacteria for approximately five weks post- procesing. (Key words: shel eggs, cool wash, egg quality) 64 INTRODUCTION Shel egg procesors who chose to produce USDA ?shielded? eggs must abide by specific USDA regulations. One such regulation states that egg wash water must be at least 90?F (32.2?C), or 20?F (11.1?C) warmer than the warmest egg entering the procesing line (7 CFR 56.76(f)(3)). Due to this regulation, eggs from in-line operations (hen houses directly connected to the procesing facility) can be washed in water as hot as 48.9?C. Research supporting the regulation was conducted by Brant and Star in 1966. In 1940, Haines and Moran observed that when eggs are placed in a bacteria suspension cooler than their internal temperature, a negative presure gradient is created, drawing bacteria through the shel and into the egg?s interior. In 1952, Lorenz and Star discovered that eggs washed in cold water were more likely to spoil than eggs washed in warm water. When this research was conducted, however, the most common way to wash eggs was by imersion washing. Eggs were placed in a wire basket, a household laundry or dish detergent was added, the basket and the eggs were submerged in water, and agitated for approximately one to thre minutes (Hutchinson et al., 2003). In 1975, imersion washing was banned and replaced by spray washing (USDA, 1975). The most recent regulation pertaining to egg procesing applies to al shel eggs and requires them to be stored in a post-procesing environment of 7.2?C or below (USDA, 1999). This regulation was established in order to decrease the amount of time needed to reduce internal egg temperatures post-procesing, and hopefully control spoilage and potential foodborne pathogens asociated with eggs. Studies have shown that due to washing, grading, and packaging, post-procesing internal egg temperatures can be 6.1 to 7.8?C higher than initial egg temperatures (Anderson et al., 1992). When 65 compared to a single wash tank, dual wash tank systems commonly used by most egg procesors mean that the shel eggs wil stay in a hot wet environment for a longer period of time (Curtis, 1999). After being procesed, eggs are typicaly packaged in pulp or foam cartons or cardboard flats, placed in cases, and paletized. In addition to the initial rise, insulation provided by packaging conditions can cause the eggs? internal temperature to continue to rise (Anderson et al., 1992). The internal temperature of packaged eggs can continue to rise for up to six hours after eggs are placed in a cooler. In fact, it may actualy take the centermost egg in a palet five to six days to reach an internal temperature of 7.2?C when stored in an environment with an ambient temperature of 7.2?C (Anderson et al., 1992; Chen et al., 2002; Jones et al., 2002b). It is important for the internal temperature of an egg to be below 7.2?C as quickly as possible because Salmonela Enteritidis (SE), the organism ost often asociated with foodborne disease and eggs, does not grow wel at refrigerated temperatures (Gast and Holt, 2000; Bel and Kyriakides, 2002). Because most eggs reach the retail outlet in such a short period of time (Bel et al., 2001; Paterson et al., 2001), reducing their internal temperature to 7.2?C or below before they are purchased by consumers can be chalenging. Maintaining the microbial integrity of the egg somewhat depends on internal egg quality. Measuring Haugh unit values (HU) and viteline membrane strength are two ways to ases an egg?s internal quality. Determining the HU is a common way to ases interior egg quality (Haugh, 1937) and has been acepted by USDA-AMS as a valid and reliable method (USDA, 2000). It is a function of egg weight and the height of the thick albumen (Haugh, 1937). The HU value, like viteline membrane strength, tends to 66 decline as the egg ages (Wiliams, 1992; Jones et al., 2002b; Jones and Musgrove, 2005; Samli et al., 2005). The viteline membrane, which surrounds the yolk, is responsible for keeping the yolk contents separate from the albumen. Determining viteline membrane strength is important because a strong viteline membrane wil prevent the yolk contents from entering the albumen. The yolk contains nutrients that are good growth medium for bacteria (Clay and Board, 1991; Humphrey and Whitehead, 1993; Gast and Holt, 2000). When the viteline membrane weakens or breaks, these nutrients can contaminate the albumen and possibly inhibit its antimicrobial properties (Clay and Board, 1991; Humphrey and Whitehead, 1993; Humphrey, 1994; Gast and Holt, 2000). As the egg ages, viteline membrane strength declines, reducing the interior quality of the egg and potentialy causing leakage of yolk nutrients or alowing bacteria to penetrate the yolk (Conner et al., 2002; Jones et al., 2002a; Chen et al., 2005). Scientists have been focusing on finding ways to reduce the egg?s internal temperature during and after procesing. They have developed methods based on the use of cryogenic gases as wel as forced cool air to rapidly cool shel eggs post-procesing (Curtis et al., 1995 and Thompson et al., 2000, respectively). Although it has been shown that egg quality is maintained or even enhanced by these methods of rapid cooling (Curtis et al., 1995; Thompson, et al., 2000; Jones et al., 2002b), the egg industry?s use of these methods has been limited due to cost and space constraints. It has been suggested that washing eggs in cool water, as opposed to warm water, would help diminish the increase in internal egg temperature during procesing. This would, in turn, aid in reaching and maintaining a post-procesing internal egg temperature of 7.2?C more rapidly without great procesing costs. 67 Previous research indicates that washing eggs in cool water could be a viable means of maintaining or enhancing egg cooling and subsequent physical and microbial quality during storage. Cooler wash water temperatures help to reduce the amount of time needed to cool eggs (Lucore et al., 1997). Cooling eggs to an internal temperature of 7.2?C and below reduces microbial contamination by inhibiting the growth of SE and other psychotropic microorganisms that may be present (Rhorer, 1991; Curtis, 1999; Bel and Kyriakides, 2002; Chen et al., 2002). Lucore et al. (1997) reported decreased bacterial counts on egg shels as wash water temperature decreased. Cool water washing of shel eggs could benefit egg procesors by initiating the cooling proces of the egg and shortening cooling time after being placed into the cooler. Other benefits of cool water washing would include a reduced cost of heating the wash water and cooling the post- procesing cooler. By commercialy procesing shel eggs at four diferent wash water temperature schemes, this study examined how cool water washing afects interior egg quality, as wel as aerobic bacterial levels and yeasts and molds on and within the egg. MATERIALS AND METHODS Egg Procesing This study was conducted in two commercial shel egg procesing facilities (A and B). At each facility, shel eggs were washed after regular procesing hours over thre consecutive days (replicates). Both facilities were operated by the same integrator, were AMS inspected, and used dual washer systems from the same manufacturer to wash eggs. In order to determine the efects of washing shel eggs in cool water, the wash water utilized in this study was collected after it had been re-circulated for four hours during the regular procesing day and contained an organic load. This created a ?worst case 68 scenario? and enabled us to beter determine the efects of cool water washing by taking into acount the recycling of wash water. The previously used wash water in each wash tank was pumped into four 55 galon drums (Consolidated Plastics Co., Inc, Twinsburg, OH). In order to prevent rust contamination, the interior of each drum was treated with a corrosive inhibitor. Once the drums were filed with the previously used wash water, they were placed in the procesing facility?s post-procesing cooler, which had an ambient temperature of approximately 7.2?C. The temperature of the wash water was then lowered to 23.9?C or slightly lower. In order to lower the wash water temperature, the drums remained in the facility?s post-procesing cooler for approximately five to twelve hours before conducting the study. Eggs were procesed using four wash water temperature schemes: H = 48.9?C, 48.9?C; HC = 48.9?C, 23.9?C; C = 23.9?C, 23.9?C; and CH = 23.9?C, 48.9?C (temperature of the first and second washer, respectively). The pH of the wash water from each plant was also monitored in order to ensure that it was maintained betwen 10 and 12 (sensION 156, Hach Co., Loveland, CO). The average wash water pH was 11.14 and 10.85 from Facility A and B, respectively. Approximately one palet of eggs for each temperature scheme was procesed in the same order (H, HC, C, CH) each day at each facility. After procesing, the eggs were packaged in new, clean pulp flats containing 30 eggs per flat. The flats were packaged in cardboard cases, and the cases were paletized. One 30-case palet (case = 30 dozen eggs, n = 10,800) was formed for each temperature scheme. As the eggs were being paletized, a DataWatch? data logger (Global Sensors, Mount Holly, NC) was placed into thre diferent cases in the palet for each wash water temperature scheme (Figure 1). Cases containing a data logger were placed on the top, in 69 the middle, and at the bottom of the palets. Al eggs were then stored at 7.2?C in the facilities? post-procesing cooler. The data loggers collected internal and external egg temperatures every thre minutes of storage for two weks post-procesing. Figure 2 shows a graphical representation of the average cooling data gathered from each procesing facility. Storage Study For ten weks post-procesing, procesed eggs were stored in an environment with an ambient temperature of approximately 7.2?C until analysis. Each wek of storage included thre replicates from each procesing facility (representing the thre consecutive days of procesing at each facility). During each wek of storage (wek 0 = wek of procesing), eggs were randomly selected to undergo testing in order to determine their internal and microbial quality. Haugh Unit. Each wek of storage, HU values were determined for the thre replicates from each procesing plant. For each replicate, HU values were determined for 18 eggs per temperature scheme (72 eggs per replicate) using the procedure described by Haugh (1937). The eggs were removed from storage and candled in order to exclude any cracked eggs. Shortly after being removed from storage, while the eggs were stil cool, HU values, along with albumen height and egg weight, were determined using an Egg Multi-Tester EMT 5200 (Robotmation Co., ltd, Tokyo, Japan). Viteline Membrane Strength. Viteline membrane strength was also determined for thre replicates per storage wek for each procesing plant. For each replicate, a 21-egg sample from each temperature scheme (84 eggs per replicate) was removed from storage. Shortly after being removed from storage, while the eggs were stil cool, viteline 70 membrane strength was determined using a Texture Technologies TA-XT2i texture analyzer (Texture Technologies, Scarsdale, NY). A texture analyzer determines viteline membrane strength using static compresion (Conner et al., 2002; Jones et al., 2002b; Kener et al., 2006). Before the asesment was conducted, al eggs were candled, and cracked eggs were excluded from testing. Each egg was individualy broken into a shalow dish and the yolk was positioned under a 1m, rounded end, stainles stel probe. Because Lyon et al. (1972) reported that the strongest section of the viteline membrane in near the chalazae, care was taken to ensure that measurements were not obtained from this area. Direct presure was applied to the yolk until the viteline membrane ruptured and the probe penetrated the yolk. Compresion measurements were made using a 5 kg load cel (calibrated using a 2 kg weight), 0.1 gram trigger force, and 3.2 m/sec test speed. Viteline membrane breaking strength was recorded as grams of force required to rupture the membrane. The force required to break the viteline membrane corresponds to its strength; a strong membrane requires more force to break. Microbial Analysis. During each wek of storage, microbial analysis was conducted for the thre replicates per procesing facility. For each replicate, 27 eggs per temperature scheme (108 eggs per rep) were removed from storage and candled. Cracked eggs were excluded from testing. Each egg was placed into a sterile plastic bag with 25mL of Buffered Peptone Water (BPW). Each bag was then gently shaken for approximately one minute. The BPW rinses for nine eggs were combined, resulting in thre sets of pooled exterior rinse samples. Thre 3M Petrifilm Aerobic Count plates and thre 3M Petrifilm Yeast & Mold Count plates per pooled sample were inoculated with 1mL each from the exterior rinse samples. Eggs were individualy removed from the plastic bags using 71 sterile tongs. In order to sanitize the exterior shel surface, each egg was briefly dipped into 95% ethyl alcohol and momentarily pased through the flame of a Bunsen burner. The eggs were then cracked, using the edge of a sterile surface, and the contents of nine eggs were placed in a sterile plastic bag. The shels of those nine eggs were also placed into a separate sterile plastic bag. This resulted in thre pooled sets of egg contents and thre pooled sets of egg shels. The shels were then gently crushed by hand once they were inside the sterile bag. Due to the use of 3M Petrifilm plates, BPW (90 mL) was added to the eg shel and content pools in acordance to 3M Petrifilm sample preparation guidelines. The sterile bags containing the egg shels and BPW were then gently shaken for approximately one minute. Thre 3M Petrifilm Aerobic Count plates and thre 3M Petrifilm Yeast & Mold Count plates per pooled sample were inoculated with 1mL of BPW from the crushed (interior) shel rinse. Because the shel membranes were not separated from the actual shel, interior shel samples include what is located betwen the inside of the shel and the shel membranes. Before thre 3M Petrifilm Aerobic Count plates and thre 3M Petrifilm Yeast & Mold Count plates per pooled sample were inoculated with 1mL of a 1:10 dilution of the egg contents, the mixture was placed in a Seward Stomacher (Seward Ltd., Norfolk, UK) and homogenized for one minute at 200 rpm. Al inoculated 3M Petrifilm Aerobic Count plates were incubated at 37?C for approximately 48 hours, and al 3M Petrifilm Yeast & Mold Count plates were incubated at 20?C for approximately five days. Presumptive colonies were then enumerated acording to manufacturer?s recommendations. 72 Statistical Analysis Previous research conducted to determine the efects of cool water washing of shel eggs has been performed in a laboratory seting (Lucore et al., 1997; Jones et al., 2005). Thus, the main purpose of this study was to determine the efects of cool water washing when conducted in a commercial seting. When conducting research in a commercial seting, rather than a controled laboratory environment, there can be many variables. In this study, the presence of these variables (facility and employee sanitation, environmental conditions, management, etc.) alowed us to more realisticaly compare cool water washing to the high temperatures currently required for egg procesing. Variables such as management, sanitation, egg age, type of procesing (in-line vs of- line), post-procesing cooler temperature, etc. were diferent at each procesing facility. Because procesing environments difered, significant facility diferences were found in the data collected. An example of these diferences can be sen in the post-procesing cooling data (Figure 1), average HU scores (Figure 3a), and average amounts of bacteria present on exterior shel surfaces (Figure 3b). As sen in Figure 1, eggs procesed at Facility A had lower average post-procesing temperatures than eggs procesed at Facility B. Figure 3a shows that, until wek four of storage, eggs procesed at Facility A had higher average HU values than those procesed at Facility B. Also, throughout ten weks of storage, eggs procesed at Facility B had more bacteria present on exterior shel surfaces than eggs procesed at Facility A. Due to confounding variables, data from both procesing facilities were combined before statistical analysis and a randomized complete block experimental design (block = procesing facility) was used to compare efects of wash water temperature scheme and extended storage. 73 Al data were analyzed using SAS (1999). Force required to rupture the viteline membrane, HU values, and albumen height were analyzed acording to the general linear model. Al aerobic bacteria, yeasts, and mold count data were also analyzed acording to the general linear model; however, the raw data was subjected to a log transformation before analysis. Because serial dilutions in BPW were prepared from al samples, bacterial counts from plates with no bacterial growth were recorded as 0.9 after log transformation. Any means that were found to be significantly diferent (P ? 0.05) were separated using the least-squared means option of the general linear model procedure. RESULTS Haugh Unit Average HU values were not significantly diferent amongst wash water temperature schemes (H = 67.5; HC = 68.0; C = 67.6; CH = 68.0). However, as sen in Table 1, there was a significant diference in average HU values betwen storage weks; at the end of ten weks of storage, average HU values had declined 14.8%. Scientists have questioned the validity of the HU as an acurate indicator of interior egg quality (Silversides et al., 1993); therefore, as an alternative method of determining interior quality, albumen height data were also analyzed. Wash water temperature scheme did not significantly afect average albumen height (H = 4.8mm; HC = 4.9mm; C = 4.9mm; CH = 4.9mm). As sen in Table 1, there were, however, significant diferences in the average albumen height over ten weks of storage. Due to storage, average albumen height decreased 23.2%. 74 Viteline Membrane Strength The average force required to rupture the viteline membrane was also not significantly afected by wash water temperature (H = 1.57g; HC = 1.55g; C = 1.57g; CH = 1.56g). Like average HU values and albumen height, viteline membrane strength also significantly decreased (20.6%) during ten weks of storage (Table 1). Microbial Analysis Wash water temperature did not significantly afect amounts of aerobic bacteria (log CFU/ml) present within shel matrixes (H = 2.98; HC = 3.07; C = 3.12; CH = 3.03). There were, however, significant temperature scheme x storage wek interactions in amounts of aerobic bacteria present on exterior shel surfaces (Figure 4) and in egg contents (Figure 5). Normal variation was observed in the overal growth trend of aerobic bacteria present on exterior shel surfaces during extended storage (Jones et al., 2004b; Jones et al., 2005). Although the amount of bacteria present decreased 37.6% by wek thre of storage (1.71 log CFU/ml versus 2.74 log CFU/ml initialy present), the most bacteria present on exterior shel surfaces (2.9 log CFU/ml) and in egg contents (3.8 log CFU/ml) during storage were recovered in wek six from eggs procesed in the H temperature scheme. Average amounts of bacteria present in egg contents significantly increased from 1.28 log CFU/ml to 3.22 log CFU/ml due to storage. During the first thre weks of storage, bacterial growth consistently remained low, and then steadily increased. There were also significant diferences in the amounts of aerobic bacteria present within the shel matrix of eggs betwen storage weks. Bacterial growth increased from 2.43 log CFU/ml to 3.39 log CFU/ml over ten weks, indicating a 39.5% increase due to storage. 75 Amounts of yeast present within the shel matrix of eggs, as wel as amounts of yeast and mold present in egg contents were not significantly diferent amongst wash water temperature schemes. There were, however, significant diferences betwen storage weks in amounts of yeast present within the shel matrix and in egg contents (Table 2). Average amounts of yeast present within the shel matrix increased 11% due to storage, and average amounts present in contents increased 5%. There were also significant wash water temperature scheme x storage interactions in amounts of mold present within the shel matrix of eggs and amounts of mold and yeast present on exterior shel surfaces. As sen in Figure 6b, there were only five occurrences of mold growth within shel matrixes during storage. Four of the five occurrences were during the first four weks of storage, and thre of the five growth occurrences were recovered from C eggs. Amounts of mold present on exterior shel surfaces increased during storage (Figure 6a). Eggs procesed in the CH temperature scheme experienced the most exterior mold growth throughout storage. There was litle variation in the average amount of yeast on exterior shel surfaces throughout storage (Figure 7). Over ten weks of storage, CH and HC eggs experienced the most yeast growth on exterior shel surfaces. Eggs procesed in the CH temperature scheme had more yeast growth than eggs procesed in the other temperature schemes shortly after procesing, as wel as during storage wek eight and ten. DISCUSION Analysis of the data collected during this study indicate that wash water temperature does not significantly afect average HU values, albumen height, viteline membrane strength, or average amounts of aerobic bacteria, yeast, and mold present 76 within the shel matrix of eggs. Wash water temperature did afect average amounts of aerobic bacteria, yeast, and mold present on exterior shel surfaces (Figures 4, 7, and 6a, respectively), average amounts of mold present within the shel matrix of eggs (Figure 6b), and average amounts of aerobic bacteria present in egg contents (Figure 5) at certain sampling times during extended storage. Diferences in microbial growth in egg contents due to the afects of wash water temperature and storage time did not afect microbial quality until approximately wek five of storage and later (Figure 5). Although significant, these diferences are of litle importance because it is beyond the average ?sel by? date of eggs. Acording to Bel et al. (2001) and Paterson et al. (2001), eggs currently procesed in the United States have an average ?sel by? date of thirty days and are actualy sold by nineten days post-procesing. Also, the expiration date for shel eggs, which indicates the maximum time frame for expected quality, cannot legaly exced forty-five days (USDA, 2000). Furthermore, when Jones et al. (2006) examined the efects of wash water temperature scheme (H, HC, and C only) on the presence of Campylobacter, Listeria, and Salmonela within eggs procesed during the current study, they isolated Campylobacter and Salmonela in shel and membrane emulsion samples during the first two weks post-procesing. No pathogens were detected within eggs after two weks post-procesing. The results of this study are consistent with those reported by Lucore et al. (1997). They reported that internal microbial counts from eggs spray washed with water as cool as 15.5?C were no diferent from internal microbial counts of eggs spray washed with 48.9?C water. In a more recent inoculation study, Hutchinson et al. (2004) found that wash and rinse water temperatures did not significantly efect surface populations of SE. 77 They also, however, reported that alowing wash and rinse water temperatures to fal below 34?C caused a detectable amount of content contamination. Although it is not clear why, it is possible that the results reported by Lucore et al. (1997), contradict the findings of Hutchinson et al. (2004) due to a diference in wash water pH, a diference in washing environment and equipment (pilot egg procesing equipment in a pilot plant versus a laboratory seting), or because the temperature of only the wash water was lowered and the rinse water temperature remained consistent with USDA guidelines (7 CFR 56.76(f)(11)). It should be noted that wash water pH is esential to the efectivenes of egg washing. Catalano and Knabel (1994) reported that maintaining wash water conditions at pH 11 or above prevents possible cross-contamination caused by recycled wash water by efectively reducing the number of SE present on egg shels and in wash water. Regardles of wash water temperature, as storage time progresed, the overal average HU values, albumen height, and viteline membrane strength significantly decreased (Table 1). These results are not surprising; other scientists have reported decreased HU values, albumen height, (Wiliams, 1992; Silversides and Scott, 2001; Jones et al., 2002b; Jones and Musgrove, 2005; Samli et al., 2005), and viteline membrane strength (Eliot and Brant, 1957; Hartung and Stadleman; 1963; Conner et al., 2002; Jones et al., 2002b; Chen et al., 2005) as a result of extended storage. Because scientists have questioned the validity of the HU as an acurate indicator of interior egg quality (Silversides et al., 1993), albumen height was measured throughout this study as an alternative means of determining egg quality. Although the HU is commonly used to measure interior quality, there are limitations asociated with HU 78 measurements. The HU is a relationship betwen egg weight and height of the thick albumen. The calculation is weighted exclusively for a 56.7g (2oz) egg (size large); which is why scientists have argued that the calculation is inacurate for eggs other than size large. More recently, scientists have reported that albumen height and the HU value equaly portray albumen quality (Silversides and Vileneuve, 1994). Analysis of data gathered in the curent study indicates the same. Maintaining interior egg quality is important because quality decline is generaly acompanied by increased microbial growth. The 2005 risk asesments of Salmonela Enteritidis in shel eggs and Salmonela spp. in egg products predicted that rapid cooling of eggs would be one of the most efective means of reducing ilneses from SE contaminated eggs (USDA, 2005). The physiological and chemical changes responsible for quality decline in eggs are acelerated by high temperatures, which is why it is important to cool eggs as quickly as possible (Romanoff and Romanoff, 1949; Kim et al., 1989; Rhorer, 1991; Chen et al., 2002; Conner et al., 2002). Data collected by Jones et al. (2006a) and the post-procesing cooling data colected during this study show that washing eggs in cool water succesfully prevents the excesive temperature increase caused by high water temperatures in dual wash tanks. Jones et al. (2006a) found that the surface temperature of shel eggs decreased when exposed to 23.9?C wash water. In the current study, eggs procesed using the C temperature scheme had the lowest average post-procesing temperatures, and eggs washed in the HH scheme had the highest. Although eggs procesed in the HC and CH temperature schemes did not have the lowest post-procesing temperatures, they cooled quicker than eggs procesed in the HH 79 scheme. By replacing the warm water from one wash tank with cool water, eggs are not exposed to as much heat during procesing and are able cool much faster than eggs procesed using only hot water. As previously discussed and expected, the decline in egg quality observed in the current study was acompanied by an increase in bacterial growth. During extended storage, average amounts of mold present on exterior shel surfaces and average amounts of yeast and aerobic bacteria present within the shel matrix and in egg contents did not follow the same downward trend as interior egg quality. Like those reported by Chen et al. (2005), our results suggest that the decline in viteline membrane strength and albumen viscosity over time increases the probability that microorganisms wil spread inside the eggs and possibly even invade the egg yolk. Despite the increase in aerobic bacteria, yeast, and mold growth observed in the current study during extended storage, acording to Jones et al. (2006), no pathogens were detected throughout the storage time in the contents of eggs procesed in the HC or C temperature scheme (Jones et al., 2006 did not collect data for eggs procesed in the CH temperature scheme). The overal results of this study suggest that washing shel eggs with cool water, while maintaining a pH of 10 to 12, has the potential to reduce internal egg temperature during and after procesing, without causing a decline in egg quality or increasing the presence of yeast, mold, and aerobic bacteria for approximately five weks post- procesing. The data collected during this study indicate that incorporating cool water into commercial shel egg procesing lowers post-procesing internal egg temperatures and alows for more rapid cooling. A more prompt reduction of internal egg temperature has the potential to enhance the physical qualities of eggs and improve their microbial 80 quality. Maintenance of egg quality factors such as viteline membrane strength and HU values combined with reducing internal egg temperature wil aid in preventing the growth of any potential pathogenic bacteria present. Excesive wash temperatures reduce profits due to the costs asociated with heating wash water and cooling eggs post-procesing (Anderson et al., 1992). Cool water washing could also provide economic benefits to the egg industry by reducing the energy needed to heat wash water, as wel as by decreasing the amount of energy needed to cool eggs folowing procesing. ACKNOWLEDGENTS This research project was funded through a cooperative agrement (Agrement number 58-6612-2-215) betwen the USDA gricultural Research Service and the National Aliance of Food Safety and Security. For help in conducting statistical analysis of these data, the author would like to thank Daryl Kuhlers and Omar Oyarzabal. The author would also like to thank Kevin Kener for his enginering asistance, James Elison, Jesica Moulton, Natasha Sanderfer, Vanesa Kretzschmar, and Alexis Davis for their technical asistance. 81 Figure 1. Data logger being placed into a case of procesed eggs. 82 6 8 10 12 14 16 18 20 22 1 2 3 4 5 6 7 8 9 10 Time Post-Processing (Days) T e m p e r a t u r e ( ? C ) HH HC CC CH Figure 2b 6 8 10 12 14 16 18 20 22 Figure 2a Figure 2. Average post-procesing cooling curves for eggs procesed at Facility A (2a) and Facility B (2b) 83 60 62 64 66 68 70 72 74 76 78 0 1 2 3 4 5 6 7 8 9 10 Storage Week Haugh Unit Value Facility A Facility B 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 CC CH HC HH Wash Water Temperature Scheme log C F U / m l PLANT A CFU PLANT B CFU Figure 3a Figure 3b Figure 3. Efects of procesing environment on average Haugh Unit values over ten weks of storage (3a) and average amounts of aerobic bacteria present on exterior shel surfaces amongst wash water temperature schemes (3b) 84 Table 1. Average Haugh unit values, albumen height, and force required to rupture the viteline membrane of eggs from combined procesing facilities for each wek of the storage Storage Week Haugh Unit Albumen Height Viteline Membrane Force (g) 0 73.8 G 5.61 H 1.75 F 1 71.8 F 5.35 G 1.72 F 2 71.1 EF 5.25 FG 1.70 F 3 69.8 DE 5.09 EF 1.56 DE 4 68.6 CD 4.94 DE 1.59 E 5 67.0 BC 4.78 CD 1.55 CDE 6 67.0 BC 4.74 CD 1.57 DE 7 65.9 B 4.63 BC 1.49 BCD 8 63.9 A 4.43 AB 1.43 AB 9 63.7 A 4.40 A 1.47 ABC 10 62.9 A 4.32 A 1.39 A SEM 0.36 0.04 0.02 A-H Means within a column with diferent leters are significantly diferent (P ? 0.05) 85 1 1 . 5 2 2 . 5 3 3 . 5 4 0 1 2 3 4 5 6 7 8 9 1 0 S t o r a g e T i m e ( w e e k ) log CFU/ml C C C H H C H H F i gur e 4 . E f e c t s of w a s h w a t e r t e m pe r a t ur e s c he m e a nd pos t - pr oc e s i ng s t or a ge t i m e o n a ve r a ge a m ount s of a e r obi c ba c t e r i a pr e s e nt on e xt e r i or s he l s ur f a c e s pr e s e nt on e xt e r i or s he l s ur f a c e s 86 1 1 . 5 2 2 . 5 3 3 . 5 4 4 . 5 0 1 2 3 4 5 6 7 8 9 1 0 S t o r a g e T i m e ( w e e k ) log CFU/ml C C C H H C H H F i gur e 5. E f e c t s of w a s h w a t e r t e m pe r a t ur e s c he m e a nd pos t - pr o c e s i ng s t or a ge t i m e on a ve r a ge a m ount s of a e r obi c b a c t e r i a pr e s e nt i n e gg c ont e nt s 87 Table 2. Average amounts of yeast present within the shel matrix (interior) and in the contents of procesed eggs for each wek of storage Yeast (log CFU/mL) Storage Week Interior Contents 0 1.00 A 1.00 A 1 1.00 A 1.00 A 2 1.03 AB 1.03 A 3 1.08 ABC 1.02 A 4 1.10 ABCD 1.12 B 5 1.12 ABCD 1.06 AB 6 1.17 CD 1.06 AB 7 1.22 D 1.06 AB 8 1.06 ABC 1.05 AB 9 1.13 BCD 1.04 AB 10 1.11 ABCD 1.05 AB SEM 0.02 0.01 A-D Means within a column with diferent leters are significantly diferent (P ? 0.05) 88 1 1 . 1 1 . 2 1 . 3 0 1 2 3 4 5 6 7 8 9 1 0 S t o r a g e T i m e ( w e e k ) log CFU/ml C C C H H C H H 1 1 . 1 1 . 2 1 . 3 F i g u r e 6 a F i g u r e 6 b F i gur e 6. E f e c t s of w a s h w a t e r t e m pe r a t ur e s c he m e a nd pos t - pr oc e s i ng s t or a ge t i m e on a ve r a ge a m ount s of m ol d pr e s e nt on e xt e r i o r s he l s ur f a c e s ( 6a ) a nd w i t hi n t he s he l m a t r i x ( 6b) o f p r oc e s e d e ggs 89 1 1.1 1.2 1.3 1.4 1.5 0 1 2 3 4 5 6 7 8 9 10 Storage Time (weeks) log C F U / m l CC CH HC HH Figure 7. Efects of wash water temperature scheme and post-procesing storage time on average amounts of yeast present on exterior shel surfaces 90 V. SUMARY AND CONCLUSIONS Previous research conducted to determine the efects of cool water washing of shel eggs has been performed in a pilot procesing plant seting (Lucore et al., 1997). Thus, the main purpose of this research was to determine the efects of cool water washing when conducted in a commercial seting. In order to do this, the best temperature, or combination of temperatures, for washing shel eggs while limiting the increase in internal egg temperature had to be identified. The afects of cool water washing on interior egg quality were acesed during phase one of this research. The second, and final, phase of this research studied the quality and microbiological efects of cool water washing when conducted in two commercial egg procesing facilities. Results of research conducted during phase one to determine the best temperature for washing shel eggs indicated that 24?C, when compared to 15.5?C, was the best cool water temperature for commercialy washing shel eggs. Thus, eggs procesed at commercial facilities during phase two of this study were procesed using a cool wash water temperature of 24?C. Analysis of egg quality data collected during phase one found no significant diferences betwen wash water temperature combinations in average Haugh unit values or viteline membrane strength, indicating that cool water washing does not afect interior egg quality. As expected, results from the storage study conducted during phase one showed a significant decline (P ? 0.05) in the average force 91 required to break the viteline membrane as storage time progresed. The average HU value also decreased due to storage. Eggs washed using the temperature combination commonly utilized by egg procesors (49?C, 49?C) had the lowest average HU values; whereas, eggs washed using temperature combinations containing only cool water temperatures (24?C, 24?C and 15.5?C, 15.5?C) had the greatest average viteline membrane strength and HU values. Jones et al. (2005) conducted a separate study of the eggs procesed during phase one in order to determine the efects of cool water washing on aerobic bacteria levels and SE contamination in inoculated eggs. Although external aerobic populations were lowest for eggs procesed using the temperature combination commonly utilized in the US (49?C, 49?C), Jones et al. (2005) concluded that al wash water temperature schemes investigated during phase one of this study were equaly capable of removing SE. Results of the research conducted during phase two also indicate that cool water washing does not negatively afect interior egg quality. Analysis of the data collected during this study discovered that wash water temperature did not significantly afect average Haugh unit values, albumen height, viteline membrane strength, or average amounts of aerobic bacteria, yeast, and mold present within the shel matrix of eggs. Wash water temperature did afect average amounts of aerobic bacteria, yeast, and mold present on exterior shel surfaces and in egg contents at certain sampling times during extended storage. Diferences in microbial growth due to the afects of wash water temperature and storage time did not afect microbial quality of the contents until approximately wek five of storage and later. Although significant, these diferences are of litle importance because it is beyond the average ?sel by? date of eggs. Acording to 92 Bel et al. (2001) and Paterson et al. (2001), eggs currently procesed in the United States have an average ?sel by? date of thirty days and are actualy sold by nineten days post-procesing. Also, the expiration date for shel eggs, which indicates the maximum time frame for expected quality, cannot legaly exced forty-five days (USDA, 2000). Furthermore, when Jones et al. (2006) examined the efects of wash water temperature scheme (H, HC, and C only) on the presence of Campylobacter, Listeria, and Salmonela within eggs procesed during the current study, they isolated Campylobacter and Salmonela in shel and membrane emulsion samples during the first two weks post- procesing. No pathogens were detected within eggs after two weks post-procesing. The results of research conducted during phase two of this study are consistent with those reported by Lucore et al. (1997). They reported that internal microbial counts from eggs spray washed with water as cool as 15.5?C were no diferent from internal microbial counts of eggs spray washed with 48.9?C water. In a more recent inoculation study conducted in a laboratory seting, Hutchinson et al. (2004) reported that wash and rinse water temperatures did not significantly efect surface populations of SE. They also, however, reported that alowing wash and rinse water temperatures to fal below 34?C caused a detectable amount of content contamination. Although it is not clear why, it is possible that the results of the present study, as wel as those reported by Lucore et al. (1997), contradict the findings of Hutchinson et al. (2004) due to a diference in wash water pH, or because the temperature of only the wash water was lowered and the rinse water temperature remained consistent with USDA guidelines (7 CFR 56.76(f)(11). It should be noted that wash water pH is esential to the efectivenes of egg washing. Catalano and Knabel (1994) reported that maintaining wash water conditions at pH 11 or 93 above prevents possible cross-contamination caused by recycled wash water by efectively reducing the number of SE present on egg shels and in wash water. Regardles of wash water temperature, as storage time progresed during phase two, the overal average Haugh unit values, albumen height, and viteline membrane strength significantly decreased. These results are not surprising; other scientists have reported decreased Haugh unit values, albumen height (Wiliams, 1992; Silversides and Scott, 2001; Jones et al., 2002b; Jones and Musgrove, 2005; Samli et al., 2005), and viteline membrane strength (Eliot and Brant, 1957; Hartung and Stadleman; 1963; Conner et al., 2002; Jones et al., 2002b; Chen et al., 2005) as a result of extended storage. As early as the mid 1900?s, scientists such as Lorenz and Star (1952) and March (1969) observed changes that occurred in washed eggs during storage. The egg industry is aware that storage causes a decline in egg quality and slowly breaks down the egg?s natural bariers, making it increasingly susceptible to bacterial entry and growth (Romanoff and Romanoff, 1949; Brooks and Taylor, 1955; Board, 1966; Humphrey, 1994; Wang and Slavik, 1998; Jones et al., 2004b). Because quality decline is generaly acompanied by increased microbial growth, maintaining interior egg quality is extremely important (Chen et al., 2005; Humphrey, 1994). Conner et al. (2002) found that the ability of SE to grow in albumen corresponds to a decline in viteline membrane strength. A weakened viteline membrane becomes permeable and may alow bacteria to enter the yolk, yolk contents to enter the albumen, or both (Humphrey, 1994; Conner et al., 2002; Chen et al., 2005). Because scientists have questioned the validity of the HU as an acurate indicator of interior egg quality (Silversides et al., 1993), albumen height was measured throughout 94 this study as an alternative means of determining egg quality. Although the HU is commonly used to measure interior quality, there are limitations asociated with HU measurements. The HU is a relationship betwen egg weight and height of the thick albumen. The calculation is weighted exclusively for a 56.7g (2oz) egg (size large); which is why scientists have argued that the calculation is inacurate for eggs other than size large. More recently, scientists have reported that albumen height and the HU value equaly portray albumen quality (Silversides and Vileneuve, 1994). Analysis of data gathered in the curent study indicates the same. As expected, the decline in egg quality during phase two of this study was acompanied by an increase in bacterial growth. As storage time progresed, average amounts of mold present on exterior shel surfaces and average amounts of yeast and aerobic bacteria present within the shel matrix and in egg contents did not follow the same downward trend as interior egg quality. Like those reported by Chen et al. (2005), our results suggest that the decline in viteline membrane strength and albumen viscosity over time increases the probability that microorganisms wil spread inside the eggs and possibly even invade the egg yolk. The increased microbial growth observed in the current study during extended storage is a good example of why expiration date for shel eggs cannot legaly exced forty-five days. Despite the increase in aerobic bacteria, yeast, and mold growth observed in the current study during extended storage, acording to Jones et al. (2006), no pathogens were detected throughout the storage time in the contents of eggs procesed in the HC or C temperature scheme (Jones et al., 2006 did not collect data for eggs procesed in the CH temperature scheme). 95 High wash water temperatures may be a factor asociated with acelerated quality decline. Recent scientific studies have shown that the maintenance of egg wash water at the regulated temperature is not sufficient to reduce bacterial levels to les than 10 5 CFU/mL (Jones et al., 2003); however, as the temperature of egg wash water rises, there is an increased risk of cuticle damage and thermal cracking (Wesley and Beane, 1967). Cuticle damage and thermal cracking provide ways for spoilage and pathogenic bacteria, especialy from the egg wash water, to enter the egg. Research conducted by Kinner and Moats (1981), Holley and Proulx (1986), and Lucore et al. (1997) suggest that wash water temperatures commonly used by most egg procesors is neither hot enough to kil microorganisms on the shel nor cool enough to inhibit their growth. Kinner and Moats (1981) found that wash water bacterial counts decreased, regardles of the temperature, when the water was at a pH of 10 and 11. Washing in warm water increases internal egg temperature and serves as an added buffer to prohibit quick cooling of the egg; thus alowing organisms on the shel, as wel as inside the egg, to continue to grow (Lucore et al., 1997). In 1955, Hilerman reported that wash water maintained at 46.1?C would increase internal egg temperatures by 0.22?C per second. Anderson et al. (1992) reported that washing, grading, and packaging can cause post-procesing internal egg temperatures to be 6.1 to 7.8?C higher than initial internal egg temperatures. Egg procesors? ability to rapidly lower post-procesing internal egg temperature is limited by current shel egg procesing technology and regulations governing wash water temperature (Anderson et al., 1992; Curtis, 1999); therefore, eggs do not cool to growth inhibiting temperatures very quickly. If present in even smal amounts, microorganisms such as SE have time to multiply as internal egg temperatures drop to 7?C, thus increasing the chances of 96 foodborne ilnes. The intended purpose of washing shel eggs in cool water is to more rapidly reduce post-procesing internal egg temperatures to a growth-inhibiting temperature of 7?C. The 2005 risk asesments of Salmonela Enteritidis in shel eggs and Salmonela spp. in egg products predicted that rapid cooling of eggs would be one of the most efective means of reducing ilneses from SE contaminated eggs. The physiological and chemical changes responsible for quality decline in eggs are also acelerated by high temperatures, which is anther reason why it is important to cool eggs as quickly as possible after procesing (Romanoff and Romanoff, 1949; Kim et al., 1989; Rhorer, 1991; Chen et al., 2002; Conner et al., 2002). The post-procesing cooling data collected during phase two of this study show that washing eggs in cool water succesfully prevents the excesive temperature increase caused by high water temperatures in dual wash tanks. By replacing the warm water from one wash tank with cool water, eggs are not exposed to as much heat during procesing and are able cool much faster than eggs procesed using only warm water temperatures. The overal results of this study suggest that washing shel eggs with cool water, while maintaining a pH of 10 to 12, has the potential to reduce internal egg temperature during and after procesing, without causing a decline in egg quality or increasing the presence of yeast, mold, and aerobic bacteria for approximately five weks post- procesing. The data collected during this study indicate that incorporating cool water into commercial shel egg procesing lowers post-procesing internal egg temperatures and alows for more rapid cooling. A more prompt reduction of internal egg temperature has the potential to enhance the physical qualities of eggs and improve their microbial quality, especialy during extended storage. Maintenance of egg quality factors such as 97 viteline membrane strength and HU values combined with reducing internal egg temperature wil aid in preventing the growth of any potential pathogenic bacteria present. 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Average egg weight for days 0, 30, and 60 109 Albumen Height 4.4 4.5 4.6 4.7 4.8 4.9 5 albumen height (mm) 49?C, 49?C 49?C, 24?C 49?C, 15.5?C 24?C, 24?C 15.5?C, 15.5?C 24?C, 15.5?C Figure 3. Sverage efects of wash water temperature combinations on egg weight over 60 days of storage. 110 APENDIX B. COMERCIAL STUDY: FACILITY A vs. FACILITY B Interior Quality Table 1. Average efects of wash water temperature scheme on Haugh unit values and viteline membrane strength for each procesing facility Haugh Unit Viteline Membrane Force (g) Temperature Scheme Facility A Facility B Facility A Facility B HH 66.8 68.3 1.51 1.63 HC 67.2 68.9 1.50 1.61 CC 67.4 67.9 1.53 1.61 CH 67.5 68.3 1.52 1.61 SEM 0.29 0.32 0.01 0.01 111 Table 2. Average efects of wash water temperature scheme on albumen height for each procesing facility Albumen Height (m) Temperature Scheme Facility A Facility B HH 4.75 4.91 HC 4.78 4.97 CC 4.83 4.89 CH 4.84 4.95 SEM 0.03 0.03 A,B Means within a column with diferent leters are significantly diferent (P ? 0.05) 112 3.5 4 4.5 5 5.5 6 0 1 2 3 4 5 6 7 8 9 10 Storage Time (week) Albumen Height (mm) Facility A Facility B Figure 1. Average efects of storage time on albumen height for each facility 113 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 0 1 2 3 4 5 6 7 8 9 10 Storage Time (week) Force (g) Facility A FacilityB Figure 2. Average efects of storage time on force required to rupture the viteline membrane of eggs from each facility 114 Aerobic Bacteria 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 CC CH HC HH Wash Water Temperature Scheme log CFU/ml PLANT A CFU PLANT B CFU Figure 3. Average efects of wash water temperature scheme on amounts of aerobic bacteria present within the shel matrix (interior) of eggs from each procesing facility 115 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 CC CH HC HH Wash Water Temperature Scheme log CFU/ml PLANT A CFU PLANT B CFU Figure 4. Average efects of wash water temperature scheme on amounts of aerobic bacteria present in the contents of eggs from each procesing facility 116 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0 1 2 3 4 5 6* 7 8 9 10 Storage Time (week) log CFU/ml FACILITY A CFU FACILITY B CFU Figure 5. Average efects of wash water temperature scheme on amounts of aerobic bacteria present within the shel matrix (interior) of eggs from each procesing facility *Data collected during storage wek 6 from eggs procesed at Facility B is mising due to technical dificulties. 117 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0 1 2 3 4 5 6* 7 8* 9 10 Storage Time (week) log CFU/ml FACILITY A CFU FACILITY B CFU Figure 6. Average efects of wash water temperature scheme on amounts of aerobic bacteria present in the contents of eggs from each procesing facility *Data collected during storage wek 6 from eggs procesed at Facility B and wek 8 from Facility A are mising due to technical dificulties. 118 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0 1 2 3 4 5 6* 7 8* 9 10 Storage Time (week) log CFU/ml FACILITY A CFU FACILITY B CFU Figure 7. Average efects of wash water temperature scheme on amounts of aerobic bacteria present on the exterior shel surface of eggs from each procesing facility *Data collected during storage wek 6 from eggs procesed at Facility B and wek 8 from Facility A are mising due to technical dificulties. 119 Yeast Table 3. Average efects of wash water temperature scheme on amounts of yeast (log CFU/ml) present on exterior shel surfaces, within the shel matrix, and in contents of eggs procesed at each facility EXTERIOR WITHIN SHEL CONTENTS Temperature Scheme Facility A Facility B Facility A Facility B Facility A Facility B HH 1.05 1.15 1.05 1.15 1.02 1.06 HC 1.05 1.19 1.04 1.14 1.02 1.07 CC 1.05 1.16 1.03 1.13 1.02 1.05 CH 1.05 1.26 1.04 1.17 1.01 1.08 SEM .01 .03 .01 .03 .01 .02 120 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0 1 2 3 4 5 6 7 8 9 10 Storage Time (week) log CFU/ml FACILITY A FACILITY B Figure 8. Average efects of wash water temperature scheme on amounts of yeast present on the exterior surface of eggs from each procesing facility 121 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0 1 2 3 4 5 6 7 8 9 10 Storage Time (week) log CFU/ml FACILITY A FACILITY B Figure 9. Average efects of wash water temperature scheme on amounts of yeast present within the shel matrix of eggs from each procesing facility 122 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0 1 2 3 4 5 6 7 8 9 10 Storage Time (week) log CFU/ml FACILITY A FACILITY B Figure 10. Average efects of wash water temperature scheme on amounts of yeast present in the contents of eggs from each procesing facility 123 Mold Table 4. Average efects of wash water temperature scheme on amounts of mold (log CFU/ml) present on exterior shel surfaces, within the shel matrix, and in contents of eggs procesed at each facility EXTERIOR WITHIN SHEL CONTENTS Temperature Scheme Facility A Facility B Facility A Facility B Facility A Facility B HH 1.04 1.02 1.0 1.0 1.0 1.0 HC 1.04 1.05 1.01 1.0 1.0 1.0 CC 1.04 1.04 1.01 1.01 1.0 1.0 CH 1.08 1.04 1.0 1.0 1.01 1.0 SEM .02 .02 .04 .05 .04 .00 124 Table 5. Average efects of storage time on amounts of mold (log CFU/ml) present on exterior shel surfaces, within the shel matrix, and in contents of eggs procesed at each facility EXTERIOR WITHIN SHEL CONTENTS Storage Time (wek) Facility A Facility B Facility A Facility B Facility A Facility B 0 1.03 1.06 1.01 1.0 1.0 1.0 1 1.0 1.07 1.0 1.01 1.01 1.0 2 1.0 1.0 1.01 1.02 1.0 1.0 3 1.02 1.0 1.0 1.0 1.0 1.0 4 1.02 1.01 1.01 1.0 1.0 1.0 5 1.01 1.06 1.0 1.0 1.01 1.0 6 1.02 1.04 1.0 1.0 1.0 1.0 7 1.04 1.03 1.0 1.0 1.0 1.0 8 1.09 1.05 1.0 1.0 1.0 1.0 9 1.13 1.06 1.0 1.01 1.0 1.0 10 1.21 1.01 1.0 1.0 1.02 1.0 SEM .03 .03 .01 .01 .01 .0