MICROBIAL ANALYSIS OF SHELLED EGGS AND  
CHEMICAL AND FUNCTIONAL ANALYSIS OF  
LIQUID EGGS 
 
Except where reference is made to the work of others, the work described in this 
dissertation is my own or was done in collaboration with my advisory committee.  This 
dissertation does not include proprietary or classified information. 
 
 
 
 
________________________________________ 
Vanessa Kristin Kretzschmar-McCluskey 
 
 
 
 
 Certificate of Approval:         
 
 
 
________________________ _______________________ 
Wallace D. Berry Patricia A Curtis, Chair 
Associate Professor  Professor 
Poultry Science Poultry Science  
 
 
 
 
________________________ _______________________  
Kenneth E. Anderson  Joe F. Pittman  
Professor Interim Dean
Poultry Science Graduate School 
 
MICROBIAL ANALYSIS OF SHELLED EGGS AND 
CHEMICAL AND FUNCTIONAL ANALYSIS OF  
LIQUID EGGS 
 
Vanessa Kristin Kretzschmar-McCluskey 
 
A Dissertation 
Submitted to 
the Graduate Faculty of 
Auburn University 
in Partial Fulfillment of the 
Requirements for the 
Degree of  
Doctor of Philosophy 
 
 
Auburn, Alabama 
December 17, 2007 
 
 iii
MICROBIAL ANALYSIS OF SHELLED EGGS AND 
CHEMICAL AND FUNCTIONAL ANALYSIS OF  
LIQUID EGGS 
 
Vanessa Kristin Kretzschmar-McCluskey 
Permission is granted to Auburn University to make copies of this dissertation at its 
discretion or upon the request of individuals or organizations at their expense. The author 
reserves all publication rites. 
 
 
 
 
 
 
______________________________ 
 Signature of Author 
 
 
 
 
______________________________ 
 Date of Graduation 
 
 iv
VITA 
 
 
Vanessa Kristin Kretzschmar-McCluskey, daughter of Paula Kretzschmar and 
wife of Mitch McCluskey, was born June 25, 1981 in Cullman, Alabama.  She graduated 
from Cleveland High School in Cleveland, Alabama in May 1999.  She attended Auburn 
University in Auburn, Alabama majoring in Poultry Science with a minor in Spanish.  
She graduated in December 2003 with a Bachelor of Science in Poultry Science degree.  
In January 2003, she entered the Graduate School at Auburn University to pursue her 
Ph.D. under the direction of Patricia A Curtis, Ph.D. 
 v
DISSERTATION ABSTRACT 
 
MICROBIAL ANALYSIS OF SHELLED EGGSAND 
CHEMICAL AND FUNCTIONAL ANALYSIS OF  
LIQUID EGGS 
 
 
 
Vanessa Kristin Kretzschmar-McCluskey 
 
Doctor of Philosophy, December 17, 2007 
(Bachelor of Science, Auburn University, 2003) 
 
 
158 Typed Pages 
 
Directed by Patricia A Curtis 
 
 
 
 
Research has shown that the incidence of Salmonella Enteritidis (SE) in 
commercially processed table eggs is approximately 1 in 20,000.  Although there has 
been great evolution of the egg industry, cases of SE infection continue to be 
problematic.  It has been shown that through environmental maintenance and proper 
processing and storage practices, the number of reported SE outbreaks has declined
 vi
Although numerous studies have been conducted outlining possible vectors and modes of 
transmission through inoculation of laying hens, there is little information regarding the 
natural occurrence of SE in shell eggs.   
From these experiments, it was concluded that hen age had a significant impact on 
the amount of microbial contamination, while hen strain had no effect.  It was also 
determined that each of the three molting treatments contained significant amounts of 
microflora, with the non-fasted treatment exhibiting 4 interior samples that were positive 
for Salmonella Braenderup over the course of the final 12-month portion.  There was no 
evidence of Salmonella Enteritidis discovered with any treatment during the course of the 
experiment, and none of the Salmonella spp. discovered in the environmental samples 
could be correlated to the species found in the shell eggs.    
Because of the concern surrounding the bacterial content of shelled eggs, over 
30% of those sold today are sold as pasteurized egg products.  Pasteurization virtually 
eliminates bacterial pathogens from eggs by holding them at a high temperature for an 
amount of time which is specific to the particular product.  However, previous research 
has shown that the pasteurization process can be detrimental to the functionality of eggs, 
causing problems for the baking industry.   
The results of these experiments concluded that the theory of pasteurization being 
detrimental to the functionality of whole egg products was not applicable to this study.  
The albumen portion of this study utilized the same treatments with the exception of the 
addition of a triethyl citrate whipping agent instead of citric acid. The results indicated 
that the addition of whipping agent to pasteurized product greatly improved functionality 
while having no effects on chemical composition.  
 vii
ACKNOWLEDGEMENTS 
 
 
 
The author would like to express sincere appreciation to her graduate committee, 
Drs. Ken Anderson and Wallace Berry, with special thanks going to Dr. Pat Curtis.  She 
would also like to thank the staff of the Auburn University Egg Quality Lab and the 
Piedmont Research Station for their assistance with these studies, as well as the Peaks of 
Excellence Program for sponsoring the funds for this research. To Mitch, you will never 
know how much you mean to me and how much I love and appreciate you.  Thank you 
for always allowing me to follow my dreams, being completely selfless and encouraging 
me to do my best.  To Alexis, Brooke, Jordan, Jess, Gina, Lindsay, Candice, Tasha, Amit, 
and Ms. Marcia, thank you all for making my graduate experience full of good memories.  
Finally, to all my fellow graduate students, look for the light at the end of the tunnel and 
never give up! 
 
This manuscript is dedicated to the author?s family, Mitch McCluskey, Paula 
Kretzschmar, Henry & Madge Kretzschmar, and the rest of the Kretzschmar clan.  
Thanks for your love, support, much needed comic relief, and for not raising me to be a 
?damn dummy?. 
 
 viii
 
Journal style used: Poultry Science 
 
Computer software used: Microsoft Office 2000? 
 SAS 9.1? 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 ix
TABLE OF CONTENTS 
 
LIST OF TABLES ...........................................................................................................  xii 
 
LIST OF FIGURES ........................................................................................................  xiii 
 
CHAPTER I.     INTRODUCTION .................................................................................... 1 
 
CHAPTER II.    LITERATURE REVIEW ........................................................................ 3 
 
Food Safety Issues in the United States .................................................................. 3 
 
Egg Related Salmonellosis ..................................................................................... 5 
 
Egg Associated Salmonella spp.  .............................................................................5 
 
General Salmonella Bacteria History ...................................................................... 7 
 
Salmonella Enterica Serovar Enteritidis ............................................................... 10 
 
Colonization in Laying Hens ................................................................................ 10 
 
Migration and Penetration of Shell Eggs .............................................................. 11 
 
The Cuticle and Pores ........................................................................................... 11 
 
Shell Membranes .................................................................................................. 14 
 
Albumen and Yolk ................................................................................................ 15 
 
Control Programs .................................................................................................. 20 
 
Molting .................................................................................................................. 28 
 
Liquid Egg Processing .......................................................................................... 35 
 
General Processes ................................................................................................. 35 
 
Whole Egg Pasteurization ..................................................................................... 38 
 
Albumen Pasteurization ........................................................................................ 41
 x
 
Functionality ......................................................................................................... 43 
 
Whole Egg Functionality ...................................................................................... 43 
 
Albumen Functionality ......................................................................................... 45  
Proximate Analysis ............................................................................................... 47 
 
Moisture ................................................................................................................ 47 
 
Fat ......................................................................................................................... 49 
 
Ash ........................................................................................................................ 50 
 
Protein ................................................................................................................... 51 
 
Ranges ................................................................................................................... 51 
 
Summary ............................................................................................................... 51 
 
CHAPTER III.     MANUSCRIPT I ................................................................................. 54 
 
INFLUENCE OF HEN AGE ON SHELL EGG EXTERIOR, INTERIOR, AND 
CONTENTS MICROFLORA AND SALMONELLA PREVALENCE DURING A 
SINGLE PRODUCTION CYCLE 
 
Abstract ................................................................................................................. 54 
 
Introduction ........................................................................................................... 55 
 
Materials and Methods .......................................................................................... 57 
 
Results and Discussion ......................................................................................... 61 
 
CHAPTER IV.     MANUSCRIPT II ................................................................................ 66 
 
INFLUENCE OF HEN AGE AND MOLTING TREATMENT ON SHELL EGG 
EXTERIOR, INTERIOR, AND CONTENTS MICROFLORA AND SALMONELLA 
PREVALENCE DURIGN A SECOND PRODUCTION CYCLE 
 
Abstract ................................................................................................................. 66 
 
Introduction ........................................................................................................... 67 
 
Materials and Methods .......................................................................................... 69 
 xi
 
Results and Discussion ......................................................................................... 75 
 
 
 
CHAPTER V.      MANUSCRIPT III ............................................................................... 85 
 
EFFECTS OF PASTEURIZATION AND THE ADDITION OF CITRIC ACID ON THE 
CHEMICAL ANALYSIS AND FUNCTIONALITY OF WHOLE EGG 
 
Abstract ................................................................................................................. 85 
 
Introduction ........................................................................................................... 86 
 
Materials and Methods .......................................................................................... 89 
 
Results and Discussion ......................................................................................... 94 
 
CHAPTER VI.      MANUSCRIPT VI ........................................................................... 101 
 
EFFECTS OF TRIETHYL CITRATE WHIPPING AGENT AND PASTEURIZATION 
ON THE CHEMICAL AND FUNCTIONAL ANALYSIS OF LIQUID ALBUMEN 
 
Abstract ............................................................................................................... 101 
 
Introduction ......................................................................................................... 102 
 
Materials and Methods ........................................................................................ 105 
 
Results and Discussion ....................................................................................... 110 
 
CHAPTER VII.     SUMMARY OF RESULTS ............................................................. 114 
 
BIBLIOGRAPHY ........................................................................................................... 116 
 
APPENDICES ................................................................................................................ 130
 xii
 
LIST OF TABLES 
 
 
Table 1A     Frequency of Salmonella positives by strain, age, location ...........................64 
 
Table 1B     Salmonella positives by egg component location ..........................................84 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 xiii
LIST OF FIGURES 
 
 
Figure 1A     Aerobic Plate Counts ....................................................................................65 
 
Figure 1B     Aerobic Plate Counts ....................................................................................79 
 
Figure 2A     Aerobic Plate Counts by Molting Treatment ................................................80 
 
Figure 3A     Aerobic Plate Counts Periods 14-24 .............................................................81 
 
Figure 4A     Salmonella Frequency by Molting Treatment ..............................................82 
 
Figure 5A     Salmonella Frequency by Period ..................................................................83 
 
Figure 1C     Sponge Cake Volume ...................................................................................97 
 
Figure 2B     Sponge Cake TPA .........................................................................................98 
 
Figure 3B     Custard Bloom ..............................................................................................99 
 
Figure 4B     Custard Weep ..............................................................................................100 
 
Figure 1D     Angel Food Cake Volume ..........................................................................112 
 
Figure 2C     Angel Food Cake TPA ................................................................................113 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 1
 
I.   INTRODUCTION 
 
 Over the years, the egg industry has seen its fair share of hardships.  From 
microbial contamination issues, to bad press, the industry has been able to combat 
foodborne illness and help eggs regain their rightful place as one of the most healthy food 
choices consumers can make.  However, there are always new and innovative ways to 
make the best better, and answers to lingering questions that have plagued the industry 
for decades.  Through processes such as pasteurization, shell egg washing and grading, 
and on-farm control measures, eggs have proven to be a safe and wholesome food 
product, and their uses in other industries is limitless.   
 Egg-associated salmonellosis is one of the most important public health problems 
in the United States and Europe.  SE shows no clinical signs in the adult hen and cannot 
be observed by the naked eye in the shell egg.  Like many other species of Salmonella, 
SE can live in the intestinal tract of the hen and contaminate the egg through exposure to 
feces.  However, due to the strict government regulations adopted in the 1970?s, this type 
of transmission has become increasingly rare.  It is possible, though, that the hens ovaries 
could become contaminated and the SE passed into the shell egg before the shell is 
formed (CDC, 2005).   
 The functional properties of shell eggs are well documented.  Albumen, yolk, and 
whole egg play vital roles in creating food ingredients that consumers use on a daily 
basis.  Meringues, salad dressings, and mayonnaise utilize the operative components of 
 2
yolk and egg white.  The egg industry has recently been plagued with the problem of yolk 
contamination of the albumen.  This occurs because of a weakening of the vitelline 
membrane, which allows lipids from the yolk to diffuse into the egg white.  Research 
shows this may have an effect on functional properties such as whipping height, and may 
negatively effect the essentially lipid-free environment of the albumen.  To remedy this 
situation, researchers must first know exactly how much fat has diffused through the 
vitelline membrane.  With the use of proximate analysis measurements for calibration, it 
is possible to use rapid methods to obtain the same accurate results.
 3
 
II.  LITERATURE REVIEW 
Food Safety Issues in the United States 
 Even though the United States has one of the safest food supplies in the world, 
foodborne illnesses continue to plague society, endangering public welfare and increasing 
the need for immediate action.  Each year, foodborne infections cause millions of 
illnesses and hundreds of deaths; however, most go undiagnosed and unreported (Tauxe, 
1997).  Since the symptoms of an infection are commonly associated with many other 
illnesses, a proper diagnosis is rarely achieved.  Nausea, diarrhea, abdominal cramps, 
fever, chills, and headaches can be easily associated with influenza or the common cold. 
 It is no surprise that the epidemiology of foodborne pathogens is dynamic.  In 
recent years we have seen the emergence of new microorganisms and the re-emergence 
of old ones.  Along with these changing infections, old procedures need to be updated or 
new ones created and implemented.  What was once protocol for a certain foodborne 
illness may not be effective in treating the same illness today.  Surveillance systems such 
as FoodNet and the Public Health Laboratory Information System allow physicians and 
the Centers for Disease Control and Prevention to monitor which infections are most 
reported and the number of cases and outbreaks associated with each.   
There has been much progress in the prevention of foodborne diseases.  
Unfortunately there is no simple answer as to how total deterrence can occur.  Proper
 4
 handling, not only from a consumer standpoint but from the industry as well, may hold 
the key to a safer food supply.  It is important that all food producers make an effort to 
educate consumers about the proper handling and cooking procedures of products they 
have on the market.  However, this is not the only avenue for success.  Since very little of 
our food is processed at home, it is important to understand vehicles for the transmission 
of contamination in the chain of food production.  Traditionally the food implicated in a 
foodborne outbreak was undercooked meat, poultry or seafood, or unpasteurized milk 
(CDC, 1997).  However, foods that were once considered safe may now be hazardous if 
not properly handled.  Thirty years ago, Salmonella Enteritidis (SE) was not a well 
known foodborne microorganism, but since the early 1980?s it has become an increasing 
problem in shell eggs and egg products.  The internal contents of eggs were once 
considered safe to be consumed raw, however, the emergence of SE as a major foodborne 
pathogen, we now know that this statement is false. Foods such as french toast, lasagna, 
pies and custards can pose a threat even though they are thought to be fully cooked (St. 
Louis et al, 1988). 
 It is important to human welfare that contamination is controlled at all levels of 
food production?from the farm to the table.  Controlling foodborne pathogens 
throughout the food production chain enables producers to reassure consumers that the 
foods they are purchasing are safe and wholesome.  Although foodborne illnesses have 
continued to be a problem, the United States has made substantial progress in identifying 
and controlling pathogens.  As new technology emerges, we can expect to see innovative 
procedures and protocols pertaining to food safety becoming implemented.   
 
 5
Egg Related Salmonellosis 
 Over the past twenty years the incidence of foodborne salmonellosis has seen its 
share of rise and decline.  The United States as well as other developed countries have 
been plagued with this illness as one of the leading causes of reported food related 
diseases.  From 1988 to 1995, the number of cases of salmonellosis, excluding typhoid 
fever, varies between 40,000 and 50,000 (MMWR, 1996).  The number of SE isolations 
have shown to be increasing (6-fold increase), with the main source of infection in the 
northeastern United States (FDA/CSFAN, 2004).  However, there have been increasing 
cases in the south and west.   
 Human salmonellosis is usually contracted from the consumption of undercooked 
meat and poultry, raw eggs, and milk.  Yet, many other foods have been implicated in the 
spread of salmonella organisms.  Many times this illness occurs because of in-home or 
consumer contamination, not because the product was tainted during processing.  
Although consumers may argue that illness would not have occurred if the product was 
not initially contaminated, it is important to take into account food preparation surface 
cleanliness and personal hygiene practices, as they play key roles in the transfer of 
bacteria.   This is just one of the reasons that education on proper handling and cooking 
procedures is so vital in consumer safety.  In developed countries, human to human 
transfer is uncommon but can occur (FSRIO, 2005).  This transfer would possibly be due 
to contact with fecal material.   
 Cross-contamination is a major factor in the occurrence of salmonellosis.  Often, 
foods such as poultry and eggs are not prepared to the proper temperature and illness 
 6
results.  It is easy to blame an inferior product as the source, but handling and preparation 
methods may be the real cause.   
 Throughout the 1990?s the incidence of egg related illnesses was widely 
publicized.  Luckily, by late 1998, the number of SE related foodborne salmonellosis was 
declining.  However, many of these outbreaks were confined to the northeastern United 
States.  In 1997, the District of Colombia Bureau of Epidemiology reported a rise in the 
number of incidences of gastroenteritis.  In total, 75 people were afflicted with the illness, 
which was later discovered to have come from a brand of lasagna that was served at 
seven different events.  The lasagna had become contaminated when a mixture of raw 
shell eggs and cheese used in the dish was improperly handled.  Each batch implicated 
could be traced back to the same egg / cheese mixture.  Upon further investigation, 
researchers discovered that two egg processors had obtained their eggs from farms that 
tested positive for SE.  Because of this, the manufacturer of the lasagna switched to using 
pasteurized eggs and egg products in all of its dishes. 
 Earlier that year, contaminated raw egg whites and yolks were linked to a 
salmonellosis outbreak when members of a Girl Scout troop and their parents became ill 
after consuming tainted cheesecake.  Upon further investigation, it was determined that 
the whites and yolk were prepared in a private residence and cooked in a double boiler 
until only slightly thickened.  Although at first, this looked to be only a consumer 
handling issue, but tests of the farm that supplied the eggs revealed SE positive cultures 
in the feed, water, and feces of the laying hens.  This finding led to the depopulation of 
layers on the farm, to prevent any further outbreaks of SE.  In reality, there are two issues 
that need to be addressed.  First, cooking whites and yolks until only slightly thickened 
 7
has not been scientifically shown to eliminate SE.  Secondly, there was an obvious issue 
on the farm.  Although there was no way for the consumer to know that the eggs were 
previously SE infected, it is important to be aware of proper cooking temperatures and 
techniques.  
 By the end of the 1990?s, the number of reported cases of egg related 
salmonellosis was on the decline.  By the year 2000, SE was still the second most 
common serotype reported to the CDC through the Public Health Laboratory Information 
System, but it only accounted for 6,000 isolates, a significant decrease when compared to 
previous years.  By 2001, the CDC sited only 46 confirmed outbreaks, 28 less than the 
account a decade earlier (Department of Health and Human Services, 2003).  However, 
this decline was not enough to prove that SE outbreaks were slowing down.  The 
Department of Health and Human Services wanted desperately to see a dramatic decrease 
of 50% by the year 2005.  In 2004, the U.S. Food and Drug Administration proposed a 
list of SE preventative measures to help meet the goal.  The rule included a biosecurity 
program, provisions for procurement of chicks and pullets, record keeping, cleaning and 
disinfection, pest and rodent control, producer testing of the environment, and 
refrigerated storage of the eggs at the farm level.   
Egg Associated Salmonella spp. 
General Salmonella Bacteria History 
 Salmonella is one of the most frequently reported foodborne illnesses, not only in 
the United States, but worldwide.  It was originally discovered and named for Dr. Daniel 
Salmon over 100 years ago.  Salmonella spp. infect a wide range of hosts including 
humans and can cause diseases ranging from severe enteric fever to self-limiting 
 8
gastroenteritis that, in some individuals, can become systemic and life-threatening 
(Lacey, 1993).  These microbes are among the most ubiquitous organisms that cause 
bacterial diarrhea and can cause paratyphoid or typhoid fever, depending upon the strain.   
 The Salmonella family includes over 2,300 serotypes of microscope bacteria 
which are one celled organisms (FSIS, 1998).  It is interesting to note that if these 
microorganisms are present in food, there is no taste, smell, or overall appearance 
difference.  Also, an animal infected with Salmonella will show no clinical signs, since 
this bacteria is commonly found in the intestinal tract and do not pose an immediate 
threat to health.  However, strains that cause no symptoms in animals can and do make 
people sick, and vice versa. 
 The classification of subgroup or serovars of the Salmonella family have been 
widely discussed (Salmonella Information, 2005).  The serovars that are most important 
in pathogenicity are those of the subgroup enterica, which contains over 2000 serotypes.  
These organisms are gram-negative, rod shaped, facultative, motile by means of flagella, 
and do not form spores.  The exceptions to these rules are S. gallinarum and S. pullorum 
which are non-motile. Members of the genus Salmonella are usually catalase-positive, 
oxidase-negative organisms which ferment glucose and produce both acid and gas.  
Salmonella bacteria are within the family Enterobacteriaceae.  Many strains are capable 
of growth on a chemically defined medium and can use citrate as a carbon source.  
Typical strains ferment neither lactose nor sucrose, and with a few exceptions, produce 
an abundant amount of hydrogen sulfide.  Salmonella colonies produce characteristic 
reactions on selective media.   
 9
There are many ways to classify Salmonella species.  Epidemiologically, strains can 
be classified according to their adaptation to human and animal hosts (FSIRO, 2005).  
Group1 includes those organisms which only cause disease to humans and higher 
primates (S. Typhi and S. Paratyphi).  Group 2 contains S. dublin and S. choleraesuis, 
which cause infections in cattle and swine, but rarely infect humans.  Group 3 includes 
two of the most important strains, S. Enteritidis and S. Typhimurium.  These organisms 
transmit salmonellosis from humans to animals.  Group 3 also includes other Salmonella 
serotypes that may cause gastroenteritis. 
Salmonella organisms can also be characterized by their antigen groups.  Antigen H 
or flagellar antigen, O or somatic antigen, and Vi antigen, which are possessed by only a 
few serovars, are the three main groups.  H antigen may occur in either or both of two 
forms, phase 1 and phase 2.  It is possible for these bacteria to change from one form to 
another (Giannella, 2005).  O antigens occur on the surface of the outer membrane and 
are determined by specific sugar sequences on the cell surface (Rubin & Weinstein, 
1977).  Vi antigen is present in only a few serovars, most commonly S. Typhi.  It is a 
superficial antigen that overlies the O antigen. 
 There are many factors that influence the survival of Salmonella organisms inside 
and outside their optimal environments.  Those such as pH, temperature, redox potential, 
and available nutrients affect not only the endurance of specific organisms, but their 
growth as well.  Temperature manipulation ranks among the most important 
environmental factors affecting bacterial growth, survival, and death.   
 
 
 10
Salmonella Enterica Serovar Enteritidis 
 Egg-associated salmonellosis is one of the most important public health problems 
in the United States and Europe.  SE shows no clinical signs in the adult hen and cannot 
be observed by the naked eye in the shell egg.  Like many other species of Salmonella, 
SE can live in the intestinal tract of the hen and contaminate the egg through exposure to 
feces.  However, due to the strict government regulations adopted in the 1970?s, this type 
of transmission has become increasingly rare.  It is possible, though, that the hens ovaries 
could become contaminated and the SE passed into the shell egg before the shell is 
formed (CDC, 2005).   
   Colonization in Laying Hens 
  Understanding the mechanism that emphasizes the pathway that SE infection 
occurs in laying hens is crucial to reducing the number of outbreaks and human illnesses.  
Though many believe that the primary mode of transmission is though contamination of 
the shell egg surface as it passes through the cloaca and vagina, other suggestions such as 
contamination of the yolk, ovaries, and oviduct are also accepted as possible vectors for 
transmission.  In 1990, Gast and Beard suggested that SE can frequently migrate into 
shell egg through the upper oviduct.  However, there are not many studies that 
specifically target which part of the oviduct is more susceptible to colonization.  In 2004, 
De Buck et al conducted a study that aimed to determine if the tubular gland cells of the 
isthmus or magnum were more heavily populated.  Both sets of tissues were 
experimentally inoculated with SE ands monitored for 24 hours to determine growth 
populations and patterns.  They discovered that the isthmus had higher levels of 
intracellular SE, and in turn, concluded that the evidence from the performed assays 
 11
favored that SE bacteria would more readily invade the tubular cells of the isthmus as 
compared to those of the magnum. 
Migration and Penetration of Shell Eggs 
 Microbial contamination of the hen?s egg has become a major cause of concern 
for consumers and public health officials.  While the egg is naturally equipped with 
barriers that help keep microorganisms from penetrating into the interior shell and 
contents, it is unrealistic to believe that these defenses are not subject to failure.  Exterior 
shell contamination is not an uncommon occurrence and has led to regulations that 
require washing and disinfection of table eggs before they are considered marketable to 
the public.  On the other hand, it would be quite difficult to cleanse the interior shell, 
yolk, and albumen, making it important to ensure that motile salmonellae do not find 
their way into these components.   
The Cuticle and Pores 
  The cuticle or ?bloom? of shell eggs is a protein rich layer that is thinly deposited 
on the exterior surface of the shell.  Since the shell egg exterior has several thousand 
pores that could potentially allow bacterial penetration, the cuticle acts as a protective 
barrier against contamination.  In theory, the cuticle aids in ensuring the safety of shell 
eggs.  In reality, however, the cuticle is removed or severely degraded when the eggs are 
washed or sanitized.  A weak cuticle allows bacteria such as SE to enter the inner shell 
membranes and possibly the contents through the pores.  However, a strong cuticle will 
add both strength and texture to the shell, which adds to the exterior shell quality.   
 The removal of the cuticle during washing has been a topic of controversy for 
many years.  Although regulations for egg inspection have been in place since the 1928, 
 12
actual egg washing regulations were not strictly enforced until 1964.  During this time, 
methods for washing mass quantities of eggs, via immersion washing, were developed.  
However, in the early 1960?s outbreaks of Salmonella infection led to the implementation 
of shell egg temperature regulations.  A study done by Brant and Starr (1966) determined 
that there were specific time-temperature requirements needed to de-contaminate the 
exterior shell of the egg.  Due to the outcome of this research, the USDA established 
regulations governing immersion egg washing.  In 1970, the Egg Products Inspection Act 
was passed; giving the USDA and FDA shared responsibility of egg safety regulations.  
Per this act, the FSIS would be responsible for inspecting the egg products to ensure 
proper labeling and distribution to consumers.  AMS, on the other hand, would be in 
charge of restricted, or inedible, shell eggs (FSIS, Title 21, Chapter 15, 1031-1056).  The 
role of the FDA was to govern the safety of all foods, including shell eggs under the 
FFDCA (212 USC 30, et seq.).  However, in 1975 the use of immersion washing was 
banned in egg processing facilities. Among the reasons was the controversy of microbial 
contamination.  It was argued that the removal of the cuticle by immersion washing and 
scrubbing would allow microbes found either on the exterior shell or in the wash water, 
the ability to penetrate into the egg through the pores.  Because the cuticle provides some 
protection from microorganisms, some researchers believed that its removal would only 
help contaminate the interior portions of the egg.  It was also determined that eggs should 
be washed in water that was 20 degrees warmer than the warmest egg.  This would allow 
the interior contents to swell slightly, pushing dirt and microbes away from the egg, and 
in turn, aiding in the prevention of contamination (Clauer, 1997). 
 13
 Although recent research has shown that due to the strict regulations utilized by 
the US government stating that shell eggs processed as table eggs are safe and virtually 
free of exterior microbial contamination after washing and sanitizing, the removal of the 
cuticle is still a hot topic.  European egg processing regulations do not allow eggs to be 
washed prior to consumer distribution.  EU regulations require that eggs sold directly to 
the public, defined as "Grade A", must not be washed or cleaned before or after grading. 
Producers also cannot chill or treat eggs for preservation. Grade B eggs, the second 
classification in the EU, may only be used by the food or non-food industries and can be 
washed or unwashed.  In their view, the major disadvantage of egg washing is the 
potential damage to the physical barriers, such as the cuticle, which can occur during or 
after washing, for instance from incorrect operations, in particular washing eggs in cold 
water.  It is also argued that even if eggs were washed, it would not prevent salmonellosis 
outbreaks from SE already deposited inside the egg. 
 The pores are located on the exterior surface of the egg shell.  The average egg 
contains over 7500 pores, most of which are located on the large end of the egg.  The 
concentrated numbers of pores at the large end of the egg serve a physiological purpose 
for hatching eggs by aiding in the transfer of oxygen and carbon dioxide through the air 
cell.  Although this process is not necessary for unfertilized table eggs, the pores are a 
main area of focus when discussing interior egg quality.  As the egg ages, carbon dioxide 
escapes through the pores, resulting in a decrease of interior quality, also, as the hen 
herself ages, she will lay eggs with larger pore sizes, creating a greater chance of motile 
Salmonella spp. entering through them.  Investigators have suggested that the pores in 
 14
egg shells provide a means for entrance of microorganisms into the egg with subsequent 
spoilage as a result (Kraft et al , 1957). 
Shell Membranes 
 In the 1960?s the question of whether or not the contents were pathogen-free at 
the time of lay remained unanswered (Board, 1966).  Earlier studies concluded that the 
shell was most susceptible to microbial penetration within a very short time after lay, 
based on the assumption that the yolk and albumen contract during cooling, causing 
organisms to be sucked through the pores ( Ferdinandov, 1944). In 1964, Lifshitz et al 
determined that the inner shell membrane is a greater defense mechanism than the 
exterior membrane, not because of its chemical properties but because of its mechanical 
functions.  While these scientists discovered that the outer membrane was thicker, 
heavier, and denser than the inner membrane, it did not prove to be an effective barrier 
against microbial penetration.  They concluded that this is possibly due to the fact that the 
outer membrane is composed of a network of course fibers that allowed organisms 
through easily, while the inner membrane is composed of smaller fibers that create a 
more compact network, resulting in smaller pore size. This study did not, however, do 
any microbial analysis to support their claim and looked only at the physical properties of 
the egg.  In 1962, Hartung and Stadelman concluded that there could potentially be a lag 
time of up to 20 days between penetration of the shells and the occurrence of a large 
interior bacterial load in the inner membranes.  This was attributed to the mechanical 
structure of the shell membranes and their effectiveness in providing a mechanical barrier 
against contamination.  However, it was also noted by Brooks and Taylor (1955) that it 
could be a combination of the protective membranes and the antimicrobial properties of 
 15
the albumen.  Experiments performed a few years later indicated that the lag time is more 
of a reflection of the antimicrobials such as lysozyme, and that the shell membranes play 
only a minor role in restraining microbial contamination of the albumen (Board and 
Ayres, 1965). 
 According to Lifshitz and Baker (1964), the inner and outer shell membranes are 
50-75 ? (inner membrane) and 15-17 ? (outer membrane), each being composed of a 
network of fibers consisting of keratin and mucin, with the interstitial fluid containing 
mainly albumen (Romanoff and Romanoff, 1949).  However, as reported by Lifshitz et al 
(1964) the shell membranes do not always prove to be effective barriers against microbial 
penetration.  In a study in which these researchers looked at the effectiveness of 
microbial invasion into the albumen through seeding of the air cell, they discovered that 
in order for microorganisms to pass into the albumen, they would have to penetrate 
through the inner shell membrane, which displays a much greater resistance to 
penetration than does the outer membrane.  Subsequent investigations performed by 
Brooks (1960), Board (1964), and Board and Ayers (1965) determined that there was a 
greater possibility of invasion 4-6 days post inoculation, when large amounts of 
organisms were used and the eggs were held at a temperature of 37?C.   
Albumen and Yolk 
 It has been determined that the yolk of shell eggs is a wonderful growth medium 
for certain bacteria, and Salmonella is at the forefront of concern.  Salmonella serotype 
Enteritidis has been identified as being most commonly associated with eggs.  Therefore, 
it is important to understand the mechanism by which these organisms are penetrating the 
exterior shell and migrating into and ultimately thriving in the contents.  Studies have 
 16
shown that in order for the egg to effectively deter pathogens from entering it must 
possess a thick and even cuticular layer which will plug gas exchange pores without 
reducing their efficiency, and a multi-layered shell that is firmly attached to the 
underlying intact shell membranes (Nascimento et al, 1992).  However, Nascimento et al 
found that the cuticular layer is rarely found as a uniform, thick covering, and that the 
integrity of the external and internal membranes is questionable when bacterial loads are 
high.  In a study performed by these scientists, the relationship between shell structure 
and the movement of SE across the eggshell wall was researched.  The results concluded 
that as shell quality decreases with age, a significant increase in bacterial penetration was 
observed.  The occurrence of pitted areas, which also increased with hen age, was 
significantly correlated with penetration.  The alignment of the mammillae was also 
significantly correlated.  It was determined that no one factor could be singled out as a 
causative agent for bacterial invasion, but that the structure of the shell and membranes as 
a whole played a key role and that higher quality shells would be less susceptible to 
infection.  It has also been determined that eggs from older laying flocks are more 
susceptible to bacterial infection.  This is because, as flock or hen age increases and egg 
quality decreases, the yolks of eggs laid by those hens are placed closer to the shell in 
internal orientation.  This is due to the fact that eggs from older flocks will have thinner 
albumen?the thick albumen will become extremely thin and the thin albumen will be 
almost water-like in consistency.  Also, as this process takes place, the amount of active 
lysozyme in the albumen is decreased (Marsh, 1995).  This allows a perfect environment 
for external penetration by motile Salmonella.  As the bacteria enter through the pores, 
 17
they are not faced with the same effective barriers, and can therefore easily reach the yolk 
and rapidly multiply. 
 Egg shell quality has been a serious issue in the industry for quite some time.  
While Pseudomonas spp. have been shown to more readily migrate into the shells of poor 
quality eggs (Sauter and Petersen, 1969), they pose more of a spoilage than a food safety 
threat.  Salmonella organisms, on the other hand, are dangerous to consumers because 
they can grow well in eggs and can cause infections in people of all ages.  In a study done 
by Jones et al eggs from three control strains and one commercial laying strain were 
inoculated with either Pseudomonas flourescens, SE, or a combination of the two bacteria 
and were stored at 26?C.  After storage, air cell, contents, exterior and interior shell 
samples were analyzed.  They determined that there was more Pseudomonas found in the 
contents of the shell eggs than SE.  This is precisely the opposite of what was determined 
to be present on the exterior shells.  This study may suggest that Pseudomonas is more 
capable of penetrating the contents than is SE.  However, this study also observed the 
differences in penetration rate as it relates to hen age.  They discovered that both SE and 
Pseudomonas flourescens were more likely to contaminate the contents, air cells, and 
internal and external membranes of eggs from older birds than those of younger flocks.  It 
was also determined that differences between the strains used in this study were 
significant.  This may suggest that certain genetic strains are more capable of producing 
eggs that many be more susceptible to microbial penetration.   
 There are essentially two ways that egg contamination can occur.  The first is 
through the exterior shell and the second is transovarian.  An important fact to note is that 
most Salmonella species, including Enteritidis, are motile and can penetrate the shell and 
 18
migrate toward the yolk (Todd, 1996).  In 2001, Grijspeerdt created a transport-reaction 
type model that incorporated a diffusion mechanism and linear transport due to motility 
and growth.  The results of this simulation showed that bacterial infection of the shells of 
eggs can lead to very high concentrations in the yolk, even before the albumen is 
saturated with organisms.  This could be due to albumen being an unfavorable growth 
medium because of lack of iron content and antimicrobials such as lysozyme.  The 
concentrations of microorganism on the shell were not seen to play a key role in the 
numbers gathered in the yolk, because this model shows that SE can grow quite well on 
the outside of the shell.  However, more studies are needed to ensure that this method was 
valid in correctly reporting bacterial movement. 
 A second study conducted by Grijspeerdt et al in 2004, researched individual-
based models that would take into account the fact that contamination of SE usually 
occurs in very low numbers.  It also looked at the transovarian method of infection and 
did not consider penetration through the shell.  Several model parameters such as 
chemotaxis, growth rate, initial numbers, and linear swimming speed were used to 
determine the results.  Bacteria were placed at several different points in the egg and their 
migration toward the yolk was monitored.  The results of this experiment showed that the 
initial site of contamination was a key factor in the amount of contamination in the yolk.  
In some areas of the albumen, the number of SE cells inoculated was less than 100, 
making it difficult for many of the cells to survive.  However, the ones that were able to 
travel to the yolk not only survived, but grew very rapidly.  Chemotaxis also played an 
important role in migration.  This is the phenomenon that motile bacteria can bias their 
random walk in the presence of attractants or repellents (Armitage, 1999).  The 
 19
microorganisms in this study were attracted to the yolk, and made every effort of 
ultimately end up there.   
 It has been reported that SE may be deposited by infected hens in the yolk or 
albumen of their eggs (Gast et al, 2005).  The higher the numbers of SE in the albumen, 
the more often the egg yolks are contaminated.  This is of great concern not only to the 
egg industry in the United States, but also in Europe, where eggs are often not washed or 
refrigerated.  Studies have shown that under certain conditions, such as unrefrigerated 
temperatures, the antimicrobials in the albumen are unable to control the growth of 
certain pathogens (Braun & Fehlhaber, 1995).  SE is no exception.  As a result of the 
unanswerable questions about SE contamination in shell eggs, the Food and Drug 
Administration defined shell eggs as a potentially hazardous food, which requires 
refrigeration during storage and care in preparation and handling. 
 In a study conducted by Conner et al (2006), experiments were conducted to 
determine the growth of SE in eggs as related to storage time and temperature, and 
vitelline membrane strength.  Eggs were stored at 4, 10, 20, or 30?C for predetermined 
times and were inoculated with SE into the albumen of each egg with enumeration 
occurring 5 days after.  The trial was replicated three times including the months of 
January, April, and August.  They determined that eggs held for 9 weeks at 4?C showed 
no signs of SE growth and their vitelline membrane strength remained constant.  
However, the other storage times showed much variation, suggesting that flock age may 
have played a role.  Although the rate at which the vitelline membrane deteriorates is 
important to determining the risk of SE in shell eggs, this study suggests that 
commercially produced eggs that are held at temperatures of ?10?C for 7 weeks does not 
 20
increase the risk of SE growth. In 2000, Gast and Holt performed a similar study which 
compared different storage temperatures and inoculation levels on the growth of SE in 
experimentally inoculated eggs.  They concluded that at 25?C storage for 2-3 days at an 
inoculation dose of 150 cells, the multiplication of SE was rapid, especially when the 
yolk was penetrated.  However, at lower doses, temperatures, and storage times, SE 
contamination was less frequently observed and invasion into the albumen proved 
potentially fatal to the low dose bacteria.  In 2001, Gast and Holt discussed the role of SE 
deposition on the yolk membrane and how this related to the contamination of the yolk 
contents.  They determined that with proper refrigeration to inhibit the growth of SE, it 
was relatively infrequent and difficult for SE to contaminate the yolks.   
Control Programs 
 Foodborne salmonellosis is a major public health concern in almost every 
country, and S. enteritidis has been reported as one of the most frequent serotypes found 
in the United States.  Because of the increasing apprehension regarding the worldwide 
food supply, countries around the globe have been forced to implement regulations to 
ensure the wholesomeness of not only the products they produce, but the ones they 
import as well.  Although the presence of any species of Salmonella can be detrimental to 
many food sources, the control of SE has become a source of interest for scientists.  The 
objective of these control measures is to deliver Salmonella-free food to consumers. 
 Because of this, the United Egg Producers (UEP) developed a ?5 star? total 
quality assurance program.  The purpose of this program was to help ensure the safety of 
the nation?s egg supply and provide consumers with the assurance that the egg industry is 
doing everything possible to produce a safe and wholesome product.  This plan targets 
 21
five critical areas of poultry and egg safety.  They include poultry house cleaning and 
disinfecting, rodent and pest elimination, proper egg washing, biosecurity, and 
refrigeration.  Because cleaning and disinfecting is the first step in the removal of visible 
dirt and soil from eggs, it is listed at the top of the ?5 star? list.  By using proper steps and 
techniques, it is possible to remove from 99-99.99% of bacteria found on exterior egg 
shells (UEP, 2006).  
  If done properly, cleaning and disinfecting reduces the source and spread of 
bacteria, and will aid in the reduction of pest infestation.  There are two types of cleaning 
that can be done, wet and dry.  Wet cleaning includes the removal of all eggs, feed and 
other debris from the poultry house and the wetting down and soaking of all areas.  It also 
includes high pressure washing of all surfaces and equipment.  Dry cleaning, on the other 
hand, includes the removal of all eggs, feed, and other debris, but with the addition of 
sweeping and blowing all dusty areas.  This can include the egg conveyor belt, feed 
troughs, and dropping boards.  It is then necessary to run scraper blades to remove all 
manure and also to remove manure from the pit areas.  After proper cleaning has been 
done, it is essential to disinfect the area.  This can be done by applying the chosen 
disinfectant to surfaces as a spray foam.  If areas cannot be reached with the foam, it may 
be necessary to use fumigation to ensure that bacteria are kept under control.   
 Rodent and pest control is another area targeted by the total quality assurance 
program.  It is no surprise that volumes of research have been conducted on the 
relationship between rodent control and the spread of harmful bacteria.  Quite simply, the 
best way to ensure that contamination does not occur from pests is to keep them out of 
the production area.  This type of control can begin with a good sanitation program, but 
 22
the help of a pest control team may be needed to design a proper plan of attack for the 
specific producer.   
 The third step of the UEP?s plan is to ensure proper washing of eggs.  They 
determined that the best way to remove both visible and invisible soil and debris is to 
wash eggs at a temperature that is greater than 100?F and has a pH greater than 11.  They 
also suggest that eggs be washed as soon after lay is possible and that a proper detergent 
can be used to soften the soil for easy removal.  Wash water and sanitizers must be 
changed every 2-4 hours to prevent organic and microbiological build-up and the 
temperature, pH, and sanitizer concentration should be closely monitored.   
 For biosecurity purposes the UEP recommends that day old chicks be purchased 
from hatcheries that participate in the National Poultry Improvement Plan (NPIP) US 
Sanitation Monitoring Program.  They also recommend that egg producers request a 9-3 
NPIP form from the chick or pullet suppliers to ensure that they are in compliance, and 
that all feed ingredients be monitored for SE.  This includes, but is not limited to, the 
proper labeling of all medications, pesticides, and feed additives that could present a 
potential hazard. 
 The final area of the ?5 star? program is refrigeration.  There have been numerous 
reports to support the theory that refrigeration helps prevent bacterial contamination.  The 
UEP suggests that eggs should be refrigerated as soon as possible after washing and 
grading.  They should be maintained at a temperature of 45?F and all transportation 
trucks should maintain this temperature.  It is also important that all data recorders, 
thermometers, and recording devices be in good working order and condition.  With the 
 23
implementation of this program producers will dramatically reduce their risk of having 
contaminated eggs and products.  It is necessary, however, to have corrective action 
programs in place just in case something fails, and have records of every action 
performed as well as verification of all procedures.  Although the idea of refrigeration has 
been proven to be a sufficient way of controlling potential bacterial hazards, it is often 
difficult to regulate.  Government agencies have been unable to provide a cohesive 
strategy for regulation of refrigeration temperatures during transportation and storage, 
and often, the way eggs are processed means that they could potentially leave packing 
plants warmer than room temperature. They are washed in hot water, immediately placed 
in cartons, stacked in pallets of several dozen cartons, and then, frequently, shrink-
wrapped in plastic. These industry practices make it difficult to cool eggs sufficiently, 
especially those eggs at the center of a pallet of cartons.  
 Since Europe does not allow washing of eggs before they are sold to consumers, 
the United States and Sweden are often looked upon with scrutiny when it comes to the 
safety of the egg supply.  However, in recent years, the EU has begun to look more 
closely at these two programs in relation to their own reports of Salmonella outbreaks. 
The Swedish control of Salmonella, particularly SE, has allowed it to be one of the few 
countries who are not involved in the spread of different SE phage types.  It has been 
reported that if any SE positives are found, the entire flock is destroyed.   
 One way that the Swedish government is controlling the risk of SE is by 
monitoring the grandparent flocks.  Approximately 10 groups of grandparent flocks are 
imported into Sweden annually, and each of these flocks are brought in as day-old chicks 
 24
(Wierup et al, 1992).  When these birds arrive, they are quarantined for 15 weeks.  They 
are tested four times during this period, and any birds that arrived dead have their liver, 
yolk sac, and cecum tested for Salmonella.  It is important to note, however, that SE has 
never been isolated from imported grandparent birds (Wahlstr?m et al, 1993). 
 Another way that Sweden controls SE is by managing their laying hens.  The 
National Food Administration, the National Veterinary Institute, and the National Board 
of Agriculture implemented policies to help regulate egg producers.  In 1995, Wierup et 
al conducted a study that looked at the prevalence of S. Enteritidis in laying flocks.  The 
first isolation of SE was discovered in 1987, when nine people became ill after visiting a 
restaurant, though none of the people ingested eggs or egg products.  The second 
incidence of SE was found in 1990 during a routine visit to a slaughterhouse.  The 
contaminated flock consisted of 60,000 layers.  In 1991, the third outbreak of SE was 
reported when 167 people became ill from a flock that consisted of 19,000 birds.  It was 
surmised that improperly heat-treated eggs were the cause.  The fourth incident was 
limited to 2200 layers and was found at the control testing stage before slaughter.  No 
human illnesses were reported.  The fifth and final discovery occurred in 1993 when farm 
children became infected with SE.  It was reported that the farm consisted of 20 hens and 
the children handled them regularly.  Because of these outbreaks, the Swedish 
government implemented mandatory testing of all flocks and any flock that is found to be 
SE positive is euthanized.  However, it is important to note that there was no correlation 
found between flock size and SE contamination.   
 25
The methods of production between the US and Sweden are similar in the fact 
that both countries are taking precautionary steps such as monitoring chicks and 
grandparent flocks for SE and testing birds to ensure that they are SE negative.  Both 
countries have had similar SE contamination rates in commercial production situations, 
and with the implementation of specific control programs, both have been able to reduce 
the number of SE related foodborne illness.   
 It has become the objective of many countries to keep the incidence of Salmonella 
below 1% at the national level, and Finland is no exception.  In order to be efficient, a 
control program must reach its targets effectively and cost efficiently (Maijala et al, 
2005).  When Finland joined the European Union, they were granted special permission 
to conduct their own Salmonella testing in meat and eggs, as well follow the procedures 
set by the EU Zoonoosis Directive.  In egg production, Salmonella has never been 
detected in breeder flocks and in layer flocks, the prevalence has been well below the 1% 
target level.  The most common serotype reported in S. typhimurium, however, SE has 
only been detected in layer flocks twice, in 1995 and 1999. 
 After the first year of the implementation of the Finnish Salmonella Control 
Program (FSCP) the target of 1% was reached with ease.  This study showed that while 
the incidence of Salmonella was low in all facets of production, it could be kept under 
control or even lowered with the practice of certain control programs.  The FSCP was 
concluded to be a necessary and successful program, which had been responsible for 
protecting public health in Finland.   
 While each country has its own ideas about regulatory actions, none has been at 
the forefront of the war on Salmonella Enteritidis like the United States.  In addition to 
 26
government regulations, other actions are being taken to control the risk of SE in the US. 
Although many of these procedures are not being used commercially, they are still key 
players when it comes to future research.  An example is the use of gamma irradiation.  
Research has shown that ionizing radiation at does up to 5 kGy can sufficiently reduce 
the numbers of non-spore forming organisms in food (Parsons and Stadelman, 1957).  
More recently a study conducted by Tellez et al, clean shell eggs were inoculated on the 
exterior shell with a solution of SE phage type 13a.  The eggs were packaged into cartons 
and transported to Mexico, where they were irradiated within 2 hours of inoculation.  The 
source of irradiation was Cobalt-60 gamma irradiation at levels of 1, 2, and 3 kGy.  The 
results illustrated that at the level of 1 kGy, there was a significant reduction of SE in the 
shell and a greater reduction in the shell membranes.  When levels of 2 and 3 kGy were 
used, the bacteria were reduced to non-detectable levels.  However, while this experiment 
had good results for the microbial analysis, the effect of the irradiation process on egg 
quality and flavor was not as promising.  The results also indicated that there was a 50% 
reduction in Haugh units and that yolk color was decreased dramatically.  Researchers 
have found that not only had egg quality deteriorated during the irradiation process, but 
egg flavor was sacrificed as well.  They determined that the dramatic changes in the 
flavor of both shell eggs and egg products made them unpalatable to consumers, often 
giving the impression of off flavors and smells.  The results of these studies could 
account for the fact that there are no irradiated eggs sold in supermarkets today.  
However, this example proves that new and innovative methods for increasing egg safety 
are not readily accepted by consumers. 
 27
 Because of the increased emergence of SE in the mid-1980?s eggs and egg 
products were given a poor reputation, being labeled the primary source of infections 
(Rodrigue et al, 1990).  Because of this, the egg industry suffered a huge blow to its 
revenue as the consumption and use of eggs declined.  Researchers began working on 
procedures to enhance the microbial quality of shell eggs using ways other than those 
regulated by government.  In a study conducted in 2001 by Barbour et al, research was 
done to produce a cleaner intact shell egg that were free from SE contamination by the 
application of pasteurized dry heat.  This experiment used strains that were identified as 
very virulent by the WHO (World Health Organization).  The eggs were inoculated using 
both high and low counts of SE cells and placed in a 57?C water bath for 25 minutes, 
followed by transfer to a hot oven at 55?C for 57 minutes.  This process served as the 
pasteurization and dry heat treatment.  The interior contents were tested for SE survival.  
The results indicated that the concentration of the SE would have to be low for the 
bacteria to be completely killed.  It is likely that this would produce eggs that were free of 
other Salmonellae as well.   
 The use of natural antimicrobials has also been studied for their effectiveness in 
controlling SE in eggs.  Products such as salad dressings and appetizers made with 
uncooked eggs have been listed as foods to be eaten with caution.  However, many of 
these foods can be made with essential oils and other natural antimicrobials that may 
reduce the risk of SE infections.  Another option may be to use pasteurized eggs, but 
more research is needed to determine how the functionality of these products differs from 
shelled eggs.  Greek appetizers that were supplemented with oregano essential oils and 
lemon juice to adjust the pH, showed a significant decrease in the number of SE 
 28
organisms and even demonstrated bacterial death if the right combination of time, 
temperature, pH, and essential oil concentration was used (Koutsoumanis et al, 1999).  
However, this experiment did not detail whether the reduction and death of the 
microorganisms was due to the oregano oils or to the adjusted pH.  It is highly possible 
that the decrease in pH was enough to lower the numbers of SE.   
 Researchers are always looking for ways to control the numbers of SE in eggs.  
These studies help the egg industry explore new discoveries in the war on foodborne 
illness.  While there has been a decline in SE infections in recent years, the numbers can 
never be low enough.  It is important to find a helpful balance between government 
regulations and innovative technologies. 
Molting 
 Molting is a natural physiological process that occurs in chickens, fowl, and other 
avian species.  It allows birds a pause in egg production and permits the reproductive 
tract and plumage time to regenerate.  In commercial laying hens, this allows the body 
time to recuperate from a long laying cycle while resulting in a state of ovarian arrest 
leading to a second cycle of eggs.  Molting is of vital economic importance to the egg 
industry.  With proper procedures, it is possible to achieve a productive life from 70 to 
105 weeks of age and for an additional 25 to 30 weeks if a second molt is used (Butcher 
& Miles, 2002). This is most important because these birds are bred specifically for egg 
production and the intent of the industry is to balance a profit through increased egg 
production, with the welfare of the birds.  
 The most common method of molting is induced molting.  This is the practice 
chosen most often in the United States to synchronize the hens production cycle schedule.  
 29
Research has shown that several physiological changes occur during an induced molt.  In 
a study done by Brake and Thaxton, hens were molted at 72 weeks of age and then again 
at 104 weeks of age.  They utilized two successive seasons, winter and summer and a 
range of temperatures that varied from -6 to 10 C during the winter molt, and 18 to 35 C 
during the summer molt.  In a similar trial utilizing commercial strains of Single Comb 
White Leghorn hens (Brake & Thaxton, 1979), it was shown that the body weights of the  
molted group were significantly less than the control group at day seven through day 35.  
The weights of the ovary, oviduct, and liver were also significantly less.  The total body 
mass loss was approximately 25% with one-fourth of this loss being attributed to the 
liver, ovary, and oviduct.   
 In addition to the physiological changes that are common with an induced molting 
process, research has also shown that there is a relationship between body weight loss 
and post-molt egg production, shell quality and shell weight.  In the early 1980s Baker et 
al conducted research that evaluated the post-molt egg production and shell 
characteristics provided by two strains of Single Comb White Leghorn hens that were 
induced into molt.  In trial 1, the hens were returned to feed after a total body weight of 
24, 27, 31, and 34% had been induced.  In trial 2, feed was returned after a body weight 
loss of 20, 25, 30, and 35%.  It was shown that the egg production was at its best when a 
weight loss of 27, 30, 31, and 35% had been achieved.  The poorest egg producers were 
those who were returned to feed with 25% or less total loss.  Shell quality in the groups 
that had higher egg production showed a value equal to or greater than that of the lower 
egg producers.  It is important to note that these hens were of different strains, ages, pre-
 30
molt treatment, and were reared differently (Baker et al, 1983).  This could have played a 
significant role in the differences that were found between the groups.   
 There has been a great debate as to which method would attain the greatest 
production rate and quality of the eggs produced.  The removal of specific nutrients such 
as sodium (Whitehead and Shannon, 1974), iodine (Arrington, et al, 1967), calcium 
(Gilbert and Blair, 1975), or zinc (Creger and Scott, 1977) will induce hens into a molt.  
It has been revealed that hens fed a calcium deficient diet of less than 0.3% will cease egg 
production in 7 to 10 days (Bell and Siller, 1962).  Two experiments were conducted to 
determine if the addition of calcium during the beginning stages of the molt would 
increase the number of marketable eggs (Garlich and Parkhurst, 1982).  The results of 
this study indicated that by adding a source of supplemental calcium, shell weight, 
percent shell and shell breaking strength did not show a significant decline.  Also, the 
incidence of unmarketable shell eggs was reduced when oyster shell was available during 
the fast.  This would suggest that calcium, while still allowing a decrease in overall body 
weight, may play an important role in shell quality and other shell characteristics.  
It has also been proposed that the addition of certain water additives to hens on a 
forced molting program may increase the livability or post-molt laying performance.  
However, research has shown there is no evidence that the addition of vitamins or 
electrolytes will enhance post-molt production or mortality during molt. In a recent study, 
four groups of  both molted and non-molted hens were given were given a treatment of 
distilled water, with the addition of 0.5% lactic acid and 0.5% acetic acid for two of the 
molted treatments (Kubena et al., 2005).  The molted hens were feed restricted for 9 days, 
and seven days prior, all treatments were placed on 8 hours of light and 16 hours of 
 31
darkness per day.  At the end of the study, the results concluded that there were higher 
levels of Salmonella  Enteritidis in the crop and cecal contents in the molted group.  
However, there were no differences in the additions of the acids to the water.  These 
studies show that the application of additives to the drinking water of laying hens has no 
effect on livability, post-molt performance, or SE positive samples after molting. 
While molting remains the primary method of recycling a laying flock, other 
methodologies are becoming more prominent in the industry.  Approaches that do not use 
complete feed withdrawal are becoming more desirable (Ruzler, 1998; Park et al., 2004; 
Anderson and Havenstein, 2007).  According to Biggs et.al. (2003) non-feed removal 
techniques are not less effective in recovering post-molt egg production.  Methods for 
non-feed removal may include feeding diets high in corn gluten, wheat middlings, corn, 
or a combination of 71% wheat middlings and 23% corn (Biggs et al. 2003, 2004).  
Additional alternatives may include a low sodium diet (Whitehead and Shannon, 1974), 
10% and 15% sugar meal (Zimmerman et al., 1987), and grape pomace with added 
thyroxine (Keshavarz and Quimby, 2002).  They also exhibit a greater skeletal integrity 
(Mazzuco et al., 2003).  These results may suggest that an alternative method of molting 
may be more animal welfare friendly than the widely accepted feed withdrawal regimens.  
However, these unconventional practices have not proven to be consistent in halting egg 
production (Berry, 2003), and may in turn, cause a dramatic decrease in lay, but not allow 
a full rest period.  This could mean that the flock would not reach it full production 
potential once the molt has concluded. 
It has been noted that hens on an induced molting regimen show higher 
incidences of Salmnella  Enteritidis (Holt and Porter, 1992) or a higher susceptibility to 
 32
SE infection (Holt, 1993) when compared to hens on a non-feed removal alternative.  It is 
necessary to mention, though, that many experiments conducted for scientific research of 
SE as it relates to molting, use inoculated laying hens as their test subjects.  Because of 
this fact, it is hard to determine if the results obtained are genuine reflections of the 
industry as a whole, or simply due to high levels of inoculum or commercial settings and 
breeds (Anderson et.al, 2001).  When looking at the molting process from an unbiased 
point of view, it is necessary to take into account all options that may play a role in the 
acquired data.  Factors such as age of hen, rearing conditions, levels of inoculum, and 
strain, may be a significant issue when choosing a molting procedure that best suits the 
research being conducted.  
 No matter which method of molting is chosen, there will always be both positive 
and detrimental effects.  Critics of the molt deny that it is a necessary part of the life of a 
flock, and refute evidence to the contrary.  Each side has their arguments, but the fact of 
the matter is that molting in the egg industry is a way of life.  The primary goal is to 
generate as many eggs as possible while taking into account the welfare of the laying 
hens.  In the future methods such as nutrient restricted diets may surpass induced or 
forced molting practices that utilize feed withdrawal.  However, for now, the feed 
restriction method for molting is the technique that is most widely practiced in research 
studies as well as in the industry. 
  Molting is a natural physiological process that avian species use to aid in the 
regeneration of feathers, healing the reproductive tract, and the addition of body weight.  
Because of this process, producers are able to recycle laying flocks for many weeks past 
the first production cycle, and still maintain efficient egg production and quality.  There 
 33
are many positive aspects to molting.  According to Hester (2005), molting decreases the 
demand for chicks by 47% and reduces the need to process, render, or bury the same 
percentage of spent hens.  In 2003, Bell discussed the fact that rejuvenation of flocks 
prevents the annual euthanasia of one hundred million male chicks.  He also ascertained 
that molting improved both livability and egg quality when comparing the second 
production cycle to the first.  In 1981, Brake et al concluded that molting resulted in the 
repopulation of the thymus gland with lymphocytes, thus aiding hen health.  Another 
advantage is feather rejuvenation.  Molting allows birds to re-grow feathers lost during 
production, thus improving thermoregulation.   
 However, with all of the positive aspects molting has to offer, there are negative 
effects as well.  When feed, water, or lighting programs are changed, undoubtedly stress 
will occur in the laying flock.  The most common molting program used is one in which 
feed is withdrawn in combination with light restriction.  This can potentially lead to 
increased mortality within the first 2 weeks of the molt (Bell, 2003).  It is also noted that 
on the first day of feed withdrawal, there was increased incidence of aggression.  While 
this behavior seemed to subside within the first few days, other behaviors such as 
increased pecking, standing, and head movement were noted (Anderson et al, 2001).  
This increase could be indicative of foraging behaviors.  It has also been noted that there 
is an increase in susceptibility to SE infection and increased numbers of SE organisms 
shed in the fecal material (Holt, 1993).  This could possibly be due to the fact that in 
fasting hens, only a small number of SE bacteria need be present to cause infection (Holt 
and Porter, 1992).   
 34
 Previous studies have shown the importance of molting to the economics of the 
egg industry, and the effects that molting has on the laying hen.  However, because of 
those effects, there is some speculation that certain commonly used molting practices are 
not in the best interest of the birds.  Although inducing a molt will recycle the laying 
flock, it is also important to ensure that post molt performance is not lost.  Due to the 
ever-increasing public concern surrounding animal welfare, Keshavarz and Quimby 
(2002) conducted a study to evaluate a number of molting techniques that would be less 
stressful than 10 to 14 day feed withdrawal, while still attaining a practical sense of 
application and satisfactory post molt performance. They discovered that post molt egg 
production was highest in the treatments that had a continuous feed withdrawal and those 
who were subjected to one day of feed withdrawal followed by a diet containing 10 ppm 
thyroxine.  They did not, however, find any differences in other post production factors 
such as egg quality.  They discovered that the use of grape pomace along with the 
thyroxine treatment would yield similar results as those obtained by feed withdrawal, but 
the results did not indicate that this procedure was less stressful on the hens.    
 In 2003 and 2004, Biggs et al evaluated non-feed removal methods for molting 
programs.  They utilized 60 week old white laying hens assigned to four treatments 
including those with restricted feed for 4 or 10 days, and those with no feed removal who 
had access to 95% corn or 95% wheat middlings molt diets, which contained additional 
vitamins and minerals.  They discovered that both the feed restricted and wheat middlings 
treatments ceased to produce eggs within 8 days.  The 95% corn diet group were still 
producing eggs at a rate of 3% at day 28, proving that this was not the best method to 
completely halt egg production.  They also concluded that the hens feed the wheat 
 35
middlings diet, as well as those fasted for 10 days had higher post molt egg production 
and egg mass when compared to the 4 day fast and corn treatments.  This study showed 
that a wheat middlings diet may be a more animal friendly way of inducing molt and that 
no drastic differences were seen when compared to the feed restricted treatment.   
 In 2001, the UEP commissioned 5 universities to conduct experiments to develop 
alternative molting programs that used non-withdrawal feeding programs to molt laying 
hens.  Some of the studies conducted included those with non-nutritive additives, lower 
sodium, and low cost ingredients that would be low in energy and protein levels.  The 
results of a study done by Koelkebeck et al (2006) concluded that it was possible to molt 
hens without removing feed, and do it successfully.  They found that by feeding laying 
hens diets consisting of soybean hulls, wheat middlings, and corn they saw results that 
correlated with those produced by the feed withdrawal method.  Because of these 
findings over the 5 year period, the UEP revised their recommendations to adopt these 
procedures and stated that only non-withdrawal methods for molting would be used after 
January 1, 2006.  These guidelines apply to approximately 82% of the industry.   
 From animal welfare issues to bacterial contamination, eggs sold to consumers as 
table eggs have had to fight to regain their proper place on the list of most nutritious 
foods.  However, one must consider that not all marketed eggs are sold in-shell.  The 
liquid egg and products industry comprises a large percentage of eggs purchased.  
Restaurants, institutions, and hospitals all utilize liquid eggs to meet their needs, and 
because of this, it is important to look closely at the processes of creating liquid eggs and 
what measures are used to ensure safety. 
 
 36
Liquid Egg Processing 
General Processes 
 Although shelled table eggs make up a large portion of the egg industry, 
pasteurized egg products are in high demand.  According to the FSIS, approximately 30% 
of the 76 billion eggs consumed in 2007 were in the form of egg products, or eggs that 
have been removed from the shell and pasteurized.  From the baking industry to specialty 
products, pasteurized products are developed utilizing whole egg mixtures, as well as 
separated yolk and albumen components.  Many consumers favor these products because 
of the idea that pasteurization aids in the destruction of harmful bacteria and because they 
are perceived as ?safer? than shelled eggs.   
 There are several processes that take an egg from its shelled form to packaged 
liquid product found in grocery stores.  One of the first steps is holding.  This includes 
choosing the eggs for breakout.  Most breakers have their own flocks used to produced 
eggs for pasteurization; therefore they are able to utilize eggs produced throughout the 
life cycle of the hen.  It is interesting to note that eggs from older hens may exhibit some 
of the same quality defects as older or aged eggs.   This is important because, as the eggs 
age, the thick albumen becomes thin and watery and the vitelline membrane of the yolk 
can deteriorate and potentially pose problems with yolk breakage and contamination of 
the albumen. For this reason, egg products industry suggests that eggs be held for no 
longer than 7-10 days at refrigeration temperatures to prevent possible problems in 
breaking (AEB, 2007). This undesirable characteristic can also be observed in eggs from 
recycled hens or hens that are reaching the end of their life cycle.   
 37
 The second step in processing liquid egg products is breaking.  This is the point 
where it is determined if the egg will be separated into yolk and albumen components or 
if it will stay as a whole egg product throughout pasteurization.  In many cases, liquid 
whole egg is created by combining albumen and yolk at the customer?s desired ratio.  It is 
important to note that not all egg processing facilities have their own breaking plants.  
Some may purchase broken out eggs from other companies, only to pasteurize and 
package them at their plant.  However, it is not uncommon for an egg company to operate 
both the breaking and pasteurization facility.  Before eggs are broken out, they are 
received at the plant, washed, sanitized, and sometimes candled to remove any eggs with 
imperfections that would render them unfit for human consumption. After this initial 
inspection, whole eggs are broken out and homogenized for uniformity.  Finally the egg 
mixture is filtered to remove any residual shell fragments, membranes, and chalazae 
(AEB, 2007) and chilled before going through thermal pasteurization.   
 For pasteurization, certain specific times and temperatures must be used to 
maintain safety.  According to the Code of Federal Regulations (Title 9, Volume 2 
590.570, 2005) every portion of the product must be heated quickly to the required 
temperature and held at that respective temperature for at least the minimum holding 
time.  In 2002, a revised edition of the International Egg Pasteurization Manual was 
published in cooperation with the United Egg Association and American Egg Board.  
This publication outlines the revised guidelines for the pasteurization of whole egg, as 
well as separate egg components, based on research in egg safety and quality.  A table 
containing the USDA pasteurization requirements is listed below (Froning, et al, 2002).  
 38
These time-temperature combinations are effective in destroying Salmonella organisms at 
a level of 10
6
 to 10
7
 cells per gram of product (Stumbo, 1965). 
Liquid Egg Product Minimum Temperature (?C) Minimum Holding Time 
(min) 
Albumen (without chemicals) 56.7; 55.6 3.5; 6.2 
Whole Egg 60.0 3.5 
Whole Egg Blends 61.1; 60.0 3.5; 6.2 
Fortified Whole Egg &Blends 62.2; 61.1 3.5; 6.2 
Salted Whole Egg 63.3; 62.2 3.5; 6.2 
Sugared Whole Egg 61.1; 60.0 3.5; 6.2 
Plain Yolk 61.1; 60.0 3.5; 6.2 
Sugared Yolk 63.3; 62.2 3.5; 6.2 
Salted Yolk 63.3; 62.2 3.5; 6.2 
 
It is important to note that products may be heated to temperatures higher than those 
listed above and can be held for longer periods of time.  However, any lesser deviation 
from the required times and temperatures will render the product as not fully pasteurized 
and the egg processing company cannot guarantee safety.  All pasteurized egg products 
are monitored for the presence of Salmonella and other microorganisms and are tested 
regularly by the processor and the FSIS.   
Whole Egg Pasteurization 
 The subject of whole egg pasteurization procedures and their relationship to the 
destruction of Salmonella bacteria has been studied since the early 1940?s.  In 1946, 
Stewart et al conducted a study in which batches of Salmonella-free liquid whole egg 
 39
were artificially inoculated with 40,000 to 100,000 Salmonella organisms per mL.  They 
wanted to determine which pasteurization procedures had the greatest detrimental effects 
to the microorganisms.  Although at this time SE was not a prevalent species, Salmonella 
Pullorum had originated as the initial Salmonella species associated with eggs and egg 
products.  This particular species has been essentially eradicated in today?s industry; 
however, it posed a potential threat to the egg industry at the time of the 1964 study.   
These researchers discovered that at a temperature of 150?F most species of Salmonella, 
including Pullorum could be destroyed when held for 0.3 minutes, creating a 6-7 log 
reduction of the test organisms.  Studies such as this helped pave the way for future 
research regarding pasteurization and eggs.   
 Although the present egg pasteurization guidelines were developed in 1969 to 
eliminate the threat of Salmonella contamination, there have been many proposed 
changes that challenge the effectiveness of the original guidelines.  In 2004, Peters et al 
conducted a series of experiments that they suspected could bring about a change to the 
guidelines.  They studied transovarian infection of eggs by SE, lower pH in egg whites 
due to rapid transport, and the addition of high amounts of sugar or salt to yolk and whole 
egg products, and new egg blends that could offer possible protection from SE.  Their 
research had merit, but more research was needed to provide the most comprehensive 
information.   
 It is extremely important to maintain the functional properties of liquid whole egg 
because it is so commonly used as an ingredient in foods products.  Studies such as those 
done by Baldwin (1986) have described how the application of heat can both positively 
and negatively impact the functionality of liquid whole egg.  The challenge for the 
 40
industry is striking a balance between ensuring consumer safety and maintaining the 
functional integrity of the product.  Because of this, Dawson and Martinez-Dawson 
(1998) conducted a study that discussed what effect specific pasteurization times / 
temperatures had on the functionality of liquid whole egg products.  By using Response 
Surface Analysis (RSA), they were able to show the effects of a range of different 
pasteurization time- temperature combinations on quality and functional factors of liquid 
egg.  They discovered that when measuring cake height volume, there was more of a time 
dependence than a temperature dependence.  This is possibly due to the fact that when 
eggs are held at high temperatures for long periods of time, their proteins begin to 
denature and unfold, making them incapable of trapping the large amounts of air needed 
to create a greater cake volume.  Solids content followed this same trend, being more 
time dependent.  Viscosity, on the other hand, was found to be both time and temperature 
dependent.  This is due to the fact that the proteins began to form a gel, making them 
more viscous.     
 Many times, citric acid is added to pasteurized whole egg products.  There is little 
research discussing the reasoning behind this addition and its effect on cooked products 
in comparison to whole egg without the added citric acid.  However, there is an obvious 
decrease in pH because of the acidic characteristics and one could conclude that this 
addition combined with pasteurization, could potentially have a positive impact on 
bacterial elimination.  By definition, citric acid is a weak organic acid that is mostly 
associated with citrus fruits.  It is a natural preservative and is often found in candies and 
soft drinks that require a sour flavor.  Though it is widely used in the egg industry, there 
is not much research on the impact of this additive on the functionality of the liquid 
 41
whole eggs.  When comparing the addition of citric acid to other acidic chelating agents 
in egg products, Gossett and Baker (1981) concluded that that although a sensory panel 
could see a decrease in flavor and color acceptability with other chelating agents, they 
could not detect a significant difference between the citric acid treatment and the control.  
This could signify that citric acid is a more reasonable choice for acid addition to egg 
products because, not only does it not change the flavor or appearance of the product, it is 
a natural preservative that aids in maintaining the yellow color of the eggs, preventing 
graying or greening.   
Albumen Pasteurization 
 It is no surprise that the pasteurization of various egg components provides a safer 
product, essentially free of microbial contamination, to consumers.  However, the 
positive aspects of pasteurization are counteracted with the negative impact it has on 
functionality, especially in the baking industry.  Pasteurized liquid egg whites are used in 
many pre-packaged products and are sold in liquid form in grocery stores.  Often, 
consumers will buy such products and expect them to perform in the same manner as a 
shelled egg in their cooked foods.  However, this is not always the case.   
 One function of egg whites in food products is to produce foam.  Foam is what is 
created when air is trapped between the protein matrices when whipping is applied.  In 
shelled eggs, it is important to make sure that the eggs used for this function are produced 
from young flocks.  As hen age increases, egg quality potentially decreases.  This can 
cause the albumen component to become thin and watery, instead of thick and distinct, 
thus creating a less voluminous foam.  This aspect can be detrimental to the baking 
 42
industry, with the fact being that eggs used from older flocks may produce cakes with 
less volume (Cunningham, et al 1960). 
 Water is the primary component of egg albumen (83%), containing protein and 
only trace amounts of fats and sugars.  The main proteins are ovalbumin (54%), 
conalbumin (13%), ovamucoid (11%), lysozyme (3.5%) which also serves as a natural 
antimicrobial, globulins (8%), and ovomucin (1.5%) (Tanner, 2007).  The protein 
partially unravels and forms a good foaming agent. A foam is formed by the protein 
forming a stable film around the included air. Studies show that the best foam is created 
when the protein is only partially unraveled. Over beating egg whites destabilizes the 
foam by fully unraveling the protein molecules. The protein is elastic, so when the egg 
white is cooked, and the air expands, the white stretches then sets in the expanded 
position.  Although albumen is comprised of several different proteins, only a few are 
responsible for foam formation and stability.  According to Cotterill and Winter (1955) 
the globulins create the actual foam formation, while the ovomucin-lysozyme complex 
aids in foam stability.  Ovalbumin and conalbumin add heat-setting properties. 
 All proteins, including those in egg white, are made of long chains of amino 
acids. In a raw egg, these are raveled up in a tangled compact mass. Chemical bonds and 
interactions between the amino acids within each protein hold this mass in a specific 
shape and stop it from unraveling. As an egg cooks, the heat causes the bonds within the 
proteins to break, a process called denaturation. As these proteins strings unfold and 
entangle with other proteins, new bonds form between these amino acids and the amino 
acids of neighboring proteins, causing the texture change in a cooked egg.  When egg 
albumen is pasteurized, the proteins it encompasses are partially denatured.  When 
 43
pasteurized product is used for baking, many of the proteins needed to produce good, 
stable foam have already been partially destroyed.  This, in turn, causes the bakery 
product to be less voluminous than if it were made with unpasteurized albumen from 
shelled eggs (Sahi, 2005). 
 Both dry and liquid egg albumen may sometimes contain additives that aid in 
foaming capabilities.  A common additive is triethyl citrate or a form of citric acid.  This 
is commonly used in pasteurized albumen products that have experienced some protein 
denaturation due to heat.  Another additive is sodium lauryl sulfate, which is added to 
improve foaming properties.  Triethyl citrate is used as a high boiling solvent and 
plasticizer for vinyl resins and cellulose acetates. It is a plasticizer permitted in the field 
of food additives, food contact material, medical, and pharmaceutical products. Sodium 
lauryl sulfate is a detergent and surfactant found in many personal care products (soaps, 
shampoos, toothpaste, etc.). It is an inexpensive and very effective foamer.  These aids 
are capable of giving the product whipping properties that are comparable to, or even 
better than, the natural untreated product.  The type of additive used is dependent on the 
type of product being produced.  For instance, although not fully understood, triethyl 
citrate seems to be more preferable in liquid frozen or pasteurized whites, while sodium 
lauryl sulfate seems to work well with dried albumen. 
Functionality 
 Eggs are very useful food ingredients and they possess a wide range of properties, 
including foaming, emulsification, gelation, color, and texturization.  Egg proteins 
provide properties in confectionary products such as cakes and meringues, as well as 
improving the texture of baked goods, quiches, and pasta.  According to Hasler (2000), 
 44
eggs are one of nature?s most perfectly functional foods.  Both albumen and yolk 
components have separate functional effects, and their combined whole egg product plays 
yet another role, producing different results.   
Whole Egg Functionality 
 Although eggs provide many functional applications, one of the most common is 
the ability of eggs to coagulate or form a solid when heated (Stadelman, 1999).  Unlike 
carbohydrate gels, which solidify upon cooling, protein gels such as the ones found in 
egg custard, exhibit the gelation process upon heating.  In basic terms, a gel is an 
intermediate phase between a solid and a liquid.  However, technically, it is defined as a 
substantially diluted system which exhibits no steady state flow.  During the process of 
whole egg gelation, proteins are first denatured into an intermediate gel state, in which 
the proteins are unfolded to expose functional groups.  This progression allows for the 
formation of the protein bonding network that eventually forms the solid state gel.  
Protein gels are irreversible, meaning that upon either re-heating or cooling, they will not 
return to their former liquid state.  This is in contrast to gelatin gels, such as those seen in 
Jello? products, in which applying heat to the solid form will cause bond breakage, 
resulting in a return to the liquid state.  
 Another functional property of whole eggs is their ability to be used as both a 
leavening and humectant agent in bakery products.  Researchers often use whole eggs as 
well as separate albumen components to study their functionality in sponge and angel 
food cakes.  Although more research is needed to determine the functional effects of 
whole eggs in food products, various experiments have utilized sponge cake volume to 
determine leavening aspects.  In recent studies, it was determined that increasing hen age 
 45
demonstrated an increase in sponge cake volume.  In theory, the more volume a cake 
presents represents a better quality egg.  Although this finding is in contrast to the 
hypothesis that eggs of older hens are lower in quality than those of younger birds, the 
effect on functionality could be due to the change in egg composition in correlation with 
increasing hen age.  As hens age, the ratio of yolk to albumen differs from that of 
younger birds, showing an increase in yolk size and a decrease and thinning of the 
albumen.  However, more research is needed to determine if this compositional change 
may explain the results. 
 Whole egg also contains a yolk portion which is essential in the emulsification of 
certain egg products such as mayonnaise and hollandaise sauce, salad dressings, cream 
puffs, and even cake batter containing shortening.  Egg yolk, in itself is considered to be 
an emulsion.  Researchers have discovered that the surface active agents in egg yolk help 
to form a film around oil globules and prevent their coalescence.  This process helps to 
create a continuous phase of the emulsification and therefore a homogenous solution.  
Separate components of the egg yolk, lecithin and cholesterol also play vital yet opposite 
roles in emulsification.  The compound cholesterol seems to favor a water-in-oil 
emulsion, whereas lecithin favors an oil-in-water emulsion (Corran and Lewis, 1924).  It 
is important to note that for a stable emulsion, it is necessary to have just the right amount 
of emulsifier, because an excess will lead to instability.   
Albumen Functionality 
 Proteins are made of long chains of amino acids. The proteins in an egg white are 
globular proteins, which means that the long protein molecule is twisted and folded and 
curled up into a more or less spherical shape. A variety of weak chemical bonds keep the 
 46
protein curled up tight as it drifts placidly in the water that surrounds it.  When you beat 
raw egg whites to make a souffl? or a meringue, you incorporate air bubbles into the 
water-protein solution. Adding air bubbles to egg whites unfolds those egg proteins just 
as certainly as heating them.   
 Some amino acids are attracted to water; they?re hydrophilic, or water-loving. 
Other amino acids are repelled by water; they?re hydrophobic, or water-fearing.  Egg-
white proteins contain both hydrophilic and hydrophobic amino acids. When the protein 
is curled up, the hydrophobic amino acids are packed in the center away from the water 
and the hydrophilic ones are on the outside closer to the water. When an egg protein is up 
against an air bubble, part of that protein is exposed to air and part is still in water. The 
protein uncurls so that its water-loving parts can be immersed in the water, and its water-
fearing parts can stick into the air. Once the proteins uncurl, they bond with each other, 
just as they did when heated, creating a network that can hold the air in place. 
 The foaming properties of albumen have long been praised for their role in 
confectionary functionality.  Often, egg white foams are associated with souffl?s, 
meringues, divinity, and angel food cakes. Because of its low fat content, angel food cake 
is frequently observed as being a healthier alternative to satisfying a person?s sweet tooth.  
One of the most noticeable factors of this product is its light, airy texture, and its ability 
to produce a pleasantly sweet taste.  The lightness of texture is due to the trapping of air 
by the proteins in the process mentioned above, while the sweet taste is due to the 
addition of sugar into the batter.  However, this addition does more than just provide a 
delicious flavor.  If added properly, it will help stabilize the foam.  If sugar is added 
before foam formation occurs, it slows the denaturation of the proteins because it 
 47
competes with the other molecules for access to water.  This does not allow the proteins 
to trap the air and create a stable foam.  However, if the sugar is added to the mixture 
after the foam is formed, it helps to generate stability, by adding a touch of viscosity 
between the trapped air bubbles.  If done properly, this should produce a stable foam with 
less weep. 
 Although the addition of sugar is one way to stabilize egg white foam, the 
addition of an acid, such as cream of tartar will also produce a stabilizing effect.  The 
addition of acid in this form lowers the pH while increasing stability (Bailey, 1935; 
Barmore, 1934).  The acidic supplement alters the net charge on the proteins, bringing 
them closer to the isoelectric point, and thus reducing the electrostatic repulsions.   
Because of this, the proteins pack more closely at the air-liquid interface, creating a more 
viscous foam (Oldham, et al, 2000).  As previously mentioned, the globulins found in 
albumen are responsible for foam formation, with the ovomucin-lysozyme complex 
contributing to foam stability (Cotterill and Winter, 1955).  It has also been revealed that 
ovomucin is the protein complex that is responsible for foam stability, while in 1981, 
Johnson and Zabik stated that conalbumin, lysozyme, ovomucin, or ovomucoid alone 
have very little or no ability to form a foam, but that the interactions of these play a vital 
role in the foaming process. 
Proximate Analysis 
Moisture 
 Taking measurements such as total solids analysis and moisture content of foods 
is imperative to the success of the foods in the industry.  Moisture content can be a 
wonderful asset or a detrimental aspect of food products.  If the moisture content is too 
 48
high, many adverse side effects, such as the unwanted growth of harmful bacteria may 
occur.  However, if the moisture content is too low, you may have problems with 
staleness and poor mouthfeel.   
 There are three main forms of water in foods.  Free water exists as free water 
within the intergranular spaces and pores of the food, and has properties that are similar 
to pure water.  This type of water acts as a dispersing agent for colloids and the solvent 
for salts.  Absorbed water, on the other hand, is water that is absorbed as a mono or poly 
molecular layer on the internal and external surfaces of the solid components by 
molecular forces.  Finally, water of hydration is water that is chemically bound, such as 
lactose monohydrate.  It can also include some salts.   
 Water is the most abundant compound in eggs.  It comprises approximately 78% 
of whole egg composition and 88% of albumen composition.  Analysis of this component 
of shell eggs can give detailed information about the amount of residual solids left in a 
sample after moisture extraction.  With this knowledge, researchers can determine what 
impact bird strain and age has on solids content, and can therefore make suggestions to 
the egg industry as to what strains, ages, or combinations would best fit specific product 
needs.  In a study done by Ahn et al (1997), utilizing hens of four different commercial 
strains of similar ages and fed consistent diets, eggs were collected, pooled, 
homogenized, and tested for differences in solid content in whole egg, yolk, and white 
components.  It was determined that the total solids content of the whole egg component 
increased with hen age.  The solids content of the albumen component was greatest in the 
younger birds (28 weeks of age), while the yolk: white ratio of eggs from hens ages 55-
75 weeks was highest.  Eggs from hens 97 weeks of age performed intermediately in all 
 49
categories.  The results of this study concluded that both young and old birds produced 
eggs with low solids content, while middle-aged hens produced eggs with high solids 
content.  They determined that that it may be more beneficial for processors and 
producers to utilize young and old hens for table egg production and birds of intermediate 
age for liquid egg production. 
 The primary method of moisture extraction involves the use of a heat source and 
time to draw out any unwanted liquid.  Drying ovens provide good moisture removal in a 
controlled environment.  Many scientific supply companies are manufacturing sample 
ovens which can be set to a specified temperature, and close tightly to ensure that no heat 
is lost or no moisture is allowed in.  In experiments involving eggs and egg products, 
ovens are tempered to 60?C and samples are dried for a period of 24 hours.  This ensures 
that no moisture, especially in the thick yolk component, is left in the sample.   
Fat 
 The egg has often been considered one of nature?s most perfect foods.  It is the 
only system, outside of mammalian, which can support the self contained growth of an 
embryo.  It provides a means of respiration, a high concentration of nutrients, as well as 
protection.  However, eggs as a food source received a poor reputation several years ago 
because of their moderate fat and cholesterol content.  Although consumer perception 
caused a decline in the 1980s and 1990s of the average number of table eggs consumed, 
the tide is now changing and eggs have fallen back into the public?s good graces.  This 
can partially be contributed to the fact that experimental research has shown what food 
scientists knew all along; that eggs have a high nutritional value, contain all 9 essential 
amino acids, and are low in both calories and saturated fat. 
 50
 The average amount of total fat in a large egg whole egg (approximately 60 
grams) is approximately 5 grams.  However, the total saturated fat, the kind that has been 
linked to a higher serum cholesterol level, is only 1.6 grams.  In albumen, the total fat is 
lowered to virtually 0 grams, with only trace amounts of lipids present.  Eggs also contain 
less than 213 mg of cholesterol per egg, making eating an egg a day perfectly fine.  The 
protein: lipid ratio in eggs is the highest of any naturally occurring food, and according to 
the American Heart Association, the amount of total fat in eggs is insignificant, and they 
are large part of many weight reducing diets.  He also stated that high levels of serum 
cholesterol and a high risk of coronary heart disease have been correlated only in 
individuals who inherit high cholesterol levels from both parents.   
Ash 
 The term ?ash?, with regards to food product analysis, refers to the inorganic 
residue that remains after total incineration of the organic matter in a particular food 
product.  This method of analysis is important to food analysis because it is a good 
general indicator of product quality, can help to determine the type and distribution of 
minerals within plant products, can give an examination of food adulteration, and can 
also be indicative of the nutritional value of the food.   
The ash content of food can and will vary within the foods selected for analysis.  
For example, the freshest foods will rarely have greater than 5% ash.  Pure oils and fats 
will contain little or no ash, however dried meat products, such as dried beef can contain 
up to 11% ash on a wet weight basis.  Foods that are mostly water, such as pasteurized 
egg products, will only contain approximately 1% ash. 
 
 51
Protein 
 Proteins are abundant in all cells and are important for specific biological 
functions and cellular structure.  Amino acids, the building blocks of proteins, are linked 
together by peptide bonds.  Proteins are composed of key elements, including hydrogen, 
carbon, oxygen, nitrogen, and sulfur, with nitrogen being the most characteristic feature 
of a protein?s chemical structure.  In this sense, protein analysis, and more specifically, 
nitrogen determination, is important in food analysis.  However, protein analysis can be 
quite tricky because some food components possess similar physiochemical 
characteristics.  For example, phospholipids, nucleic acids, and amino acids may 
contribute to non-protein nitrogen, which could cause incorrect measurements in the total 
protein content.    
Ranges 
 Before performing any method of proximate analysis, it is important to 
understand what range of values is expected for eggs as to compare your results with the 
published data.  For example, liquid or frozen albumen should contain approximately 
12.1 grams solids, 10.2 grams protein, little to no lipids, and 0.68 grams ash per a 100 
gram sample.  Along the same lines as albumen, liquid or frozen whole egg should 
contain approximately 24.5 grams total solids, 12.0 grams protein, 10.9 grams total lipids, 
and 1.00 grams ash per a 100 gram sample. 
Summary 
 There are many key components to helping the egg industry answer ?how? and 
?why? questions.  Although many studies have been conducted in the area of whipping 
performance and functionality, the problem continues to plague the industry.  Factors 
 52
such as pressure, yolk contamination, freshness and processing temperatures negatively 
affect foaming properties. It is known that phosvitin and lipovitelline phospholipids 
wreak havoc whipping performance.  This is a vital reason to keep unwanted elements 
out of egg whites. By comparing the whipping height data of unprocessed eggs to that of 
pasteurized albumen, conclusions may be drawn pertaining to foam stability and 
whipping peak height. Furthermore, findings suggest that as hen age increases, foaming 
quality and stability decreases.  It is necessary to find a way to keep consistency at a 
maximum and utilize eggs rendered to the breaker.  If eggs from young and old flocks are 
blended, it is possible to maintain an acceptable foam height and stability.  Produced 
outcomes should include added angel food cake volumes and taller, more structured 
meringues.  With further research investigators can help bridge the gap between 
information previously gained and that yet to be discovered. 
 The use of proximate analysis to calibrate cutting edge equipment is essential in 
the proper performance of such machines. Drying of the liquid portions creates the solid 
forms of albumen, whole egg, and yolk.  As albumen is heated, for example, the protein 
is denatured building a solid matrix.  When egg whites are added to tender products, it 
renders them easier to package and more convenient and palatable for the consumer. 
Comparisons of data show evidence of significant differences between unprocessed and 
pasteurized eggs.  It is widely known that pasteurization will negatively impact the 
proteins in both whole egg and albumen that contribute creating and stabilizing foams.  
Although this was once thought to be detrimental to the baking industry, the utilization of 
certain additives such as sodium lauryl sulfate and triethyl citrate are heralded with aiding 
albumen products perform almost better than before they were treated.  Although 
 53
whipping agent additives are not commonly added to whole egg products, the use of 
citric acid in pasteurized whole egg will aid in the preservation of the product, but further 
research is needed to determine what effects this additive has on the functionality of 
pasteurized whole egg.  Further inspection of these observations will pinpoint where the 
disparities occur.   
 As previously mentioned, further investigation is needed to create fundamental 
links to pertinent industry questions.  Because even trace amounts of yolk contamination 
can be detrimental to many aspects of functionality, it is imperative to determine rapid 
and accurate methods to determine the slightest amounts.  Future funding will play a 
critical role in problem solving, not only for researchers, but for the egg industry as well.   
 Pasteurization is used as a way to create a seemingly sterile egg product, free 
from pathogens, which consumers view as safe.  It is important to determine how these 
pathogen-free products perform functionally in food products.  Does pasteurization have 
an effect on the performance of whole egg both with and without the addition of citric 
acid?  How does this compare with the performance of unpasteurized product?  Is the 
chemical make-up the same or altered?  Do the same factors hold true for albumen 
products both with and without an added whipping agent?  Each of these questions is 
pertinent to the egg industry.  A look the different chemical and functional aspects of egg 
products, as well as related microbial testing may provide the needed answers.
 54
MANUSCRIPT I 
 
INFLUENCE OF HEN AGE ON SHELL EGG EXTERIOR, INTERIOR, AND 
CONTENTS MICROFLORA AND SALMONELLA PREVALENCE DURING A 
SINGLE PRODUCTION CYCLE 
 
ABSTRACT  
Salmonella Enteritidis (SE) infection in humans has been chiefly attributed to the 
consumption of raw or undercooked eggs or egg products.  The objective of this study 
was to determine if increasing hen age influenced the total microflora counts or the 
prevalence of Salmonella spp. on the egg shell surface, within the shell, or in the 
contents.  Eggs from Hy-Line W-98 and Bovans White layer strains were sampled 
approximately every 28 d from 17 to 66 wk of age from the 35
th
 North Carolina Layer 
Performance and Management (NCLP&M) Test.  The layers were managed under 
identical husbandry practices.  This study utilized 45 eggs per hen population for a total 
of 90 eggs per period. Five replicates from each strain were represented within the 
samples, with the same hens being sampled each period.  Pooled sets of 9 eggs were used 
for each strain.  The exterior, interior shell, and contents were enumerated to obtain total 
aerobic counts and tested for the prevalence of SE.   
There were 120 total egg pools taken, and, of these, there was one positive 
Salmonella sample for exterior and one for interior.  For this study, essentially all eggs 
produced by the two strains during the 35
th
 NCLP&M test were tested. Sampling of the 
egg began with rinsing the exterior shell to collect any microorganisms that may have 
 55
adhered to the surface.  The pooled rinsate accounted for the exterior counts.  The 
sampling of the interior shell included the shell membranes and matrix, as well as the 
sterilized exterior portion of the shell.  Contents sampling included both the yolk and 
albumen components, as well as the chalazae.  Hen age significantly (P < 0.05) affected 
the microbial loads on the egg components tested.  Exterior counts increased in period 4.  
Interior counts were erratic (P < 0.05), increasing  2 log units over the 12-mo cycle. 
Contents data were not significantly different (P < 0.05) until period 12, when microbial 
loads increased from 0 to 1 log unit.    
INTRODUCTION 
Microbial contamination of the hen?s egg is an ongoing concern for consumers 
and public health officials.  While the egg is naturally equipped with barriers that help 
keep microorganisms from penetrating into the interior shell and contents, it has been 
shown that these barriers can fail.   In 1964, Lifshitz and Baker determined that because 
of its density, the inner shell membrane is a greater defense mechanism than the exterior 
membrane, rather than because of its chemical properties.  While these scientists 
determined that the outer membrane was thicker, heavier, and denser than the inner 
membrane, it proved to be an ineffective barrier against microbial penetration.  Lifshitz 
and Baker (1964) also concluded that the outer membrane is composed of a network of 
course fibers that would allow organisms through easily, while the inner membrane is 
composed of smaller fibers creating a compact network, resulting in an effectively 
smaller pore size.  This study did not, however, do any microbial analysis to support their 
claim and looked only at the physical properties of the egg.  Board (1966) concluded that 
the shell was most susceptible to microbial penetration within a very short time after lay, 
 56
based on the assumption that the yolk and albumen contract during cooling, causing 
organisms to be sucked through the pores. However, he did not answer the question of 
whether or not the contents were pathogen-free at the time of lay. 
It has been known that the yolk of shell eggs is a wonderful growth medium for 
certain bacteria, and Salmonella is at the forefront of concern.  The Salmonella serotype 
Enteritidis (SE) has been commonly associated with eggs.  Therefore understanding the 
mechanisms used by these organisms to penetrate the shell and colonize contents is 
extremely important.  Nascimento (1990) found that the cuticular layer is rarely a 
uniform, thick covering and that the integrity of the external and internal membranes is 
questionable when bacterial loads are high.  It was determined that the relationship 
between shell structure and the movement of SE across the eggshell wall was important.  
It was concluded that as shell quality decreases with hen age, a significant increase in 
bacterial penetration was observed that coincided with the occurrence of pitted areas, and 
was significantly correlated with the penetration.  The alignment of the mammillae was 
also found to significantly correlate.  It was concluded that no one factor could be singled 
out as a causative agent for bacterial invasion, but rather the structure of the shell 
membranes as a whole played a combined role and that higher quality shell would be less 
susceptible to infection.  However, Nascimento et al. (1992) showed that a thick and even 
cuticular layer plugs the gas exchange pores without reducing their efficiency, and a 
multi-layered shell that is firmly attached to the underlying intact shell membranes was 
essential for the egg to effectively deter pathogens from penetrating the shell.  In 2005, 
Jones and Musgrove examined clean eggs from an in-line operation which they 
inoculated by dipping into a solution of nalidixic-resistant SE, then measured for shell 
 57
strength and external and contents contamination.  They showed only a weak negative 
correlation (r = -0.13) between external contamination of SE and shell strength, and no 
significant correlation was found between SE contamination in the contents and shell 
strength.      
MATERIALS AND METHODS 
Eggs from two commercial layer strains, Hy-Line W-98 (HL) and Bovans White 
(BW) were used as the source for monitoring the microbial population on and in shell 
eggs.  The hens were managed under the guidelines outlined in the 35
th
 North Carolina 
Layer Performance and Management (NCLP&M) Test (Anderson, 2005), under 
identical husbandry practices.  Every 28 d, the previous 24 h  egg production was 
collected from specific replicates, by staff wearing  laboratory gloves, hairnets, and 
jumpsuits,  to eliminate potential contamination by human hands, then placed in clean 
paper pulp flats and packed in a sterile plastic bag and then placed into a 30 dozen case.  
The eggs were stored in a 4?C walk-in cooler until they could be picked up from the 
facility by an Auburn University technician the same day, and brought back to the 
laboratory where they were stored overnight in a 4?C cooler before undergoing 
microbiological analysis the following day. 
Eggs for this experiment were collected from 3 replicates representing the HL and 
BW strains, with 20 eggs collected from each of the replicates.  This was done to 
compensate for any eggs which may have become cracked during transport to the 
laboratory.  Fifteen eggs from each replicate (45 eggs per respective strain) were 
randomized and the set of 45 eggs was broken down into groups of 9 egg pools for a total 
of 5 pooled sets per strain.  Each individual egg was then placed into a small, sterile 
 58
rinsate bag with the addition of 25 mL of Buffered Peptone Water (BPW; Neogen Corp., 
Acumedia, Lansing, MI).  The individual bags were then closed and shaken vigorously 
by hand for 1 min to ensure that the exterior shells were rinsed thoroughly.  Rinsates 
were then pooled into another sterile bag with 9 rinses per pool.  A small amount of 
rinsate was placed into sterile spiral plater cups
 
and plated onto Plate Count Agar (PCA; 
Neogen Corp.) using a WASP spiral plater (Microbiology International, Frederick, MD).  
The additional rinsate was incubated at 37?C for 24 h for further Salmonella testing. 
The eggs were then removed from the original rinsate bag with sterile tongs and 
flamed by dipping in 70% ethanol and passing through a Bunsen burner.  Eggs were 
cracked onto sterile foil and the contents (both yolk and albumen) placed into a sterile 
stomacher bag, while the shells were placed into a sterile filter bag and gently crushed by 
hand.  Pooled sets of 9 contents and shells were used just as with the exterior rinsates 
described above.  The contents were placed in a stomacher bag and then homogenized in 
a Seward? 400 Circulator stomacher (Seward, Norfolk, UK) at 200 rpm for 1 min, and 
10 mL was extracted and placed into another sterile stomacher bag with the addition of 
90 mL of BPW to make a 1:10 dilution.  The 100 mL of contents / BPW was then 
homogenized in a stomacher for 1 min at 200 rpm.  A small amount of the contents was 
placed into a sterile spiral plater cup to be spiral plated onto PCA.  The remaining 
mixture was placed into an incubator at 37?C for 24 h for further Samonella testing. 
The bag containing the shells included the addition of 90 mL of BPW and was 
shaken by hand for 1 min to ensure proper washing of the interior shell and membranes.  
A small amount of this rinse was also placed into a sterile spiral plate cup for spiral 
plating on PCA and the remaining rinse was incubated for 24 h at 37?C incubator for 
 59
Salmonella testing.  All spiral plated PCA plates from each of the three egg components 
(exterior rinse, contents, and interior rinse) were incubated at 37?C for 24 h to determine 
total aerobic counts.   
After a 24 h incubation period, each set of 9 pooled rinses or contents for each of 
the three components was removed from the incubator and shaken by hand for one min to 
homogenize.  Then 0.1 mL was removed from each bag, and placed into 9.9 mL of 
Rappaport-Vassiliadis R10 (RV; Neogen Corp) enrichment broth.  Once inoculated, the 
RV tubes were then placed into a 42?C incubator for 24 h.  The remaining egg rinses and 
contents were then discarded.  The PCA plates that were inoculated the day before were 
removed from the incubator and read using a QCount? plate counter (Spiral Biotech, 
Inc., Norwood, MA), using the spiral plate function. 
The RV tubes were removed from the 42?C incubator after 24 h and vortexed to 
ensure proper mixing.  A 0.1 mL bioloop
 
of inoculum was removed from the tube and 
streaked for isolation onto Brilliant Green Sulfapyridine agar (BGS; Neogen Corp.).  
Plates were then placed into a 37?C incubator for 24 h for the detection of Salmonella 
and Salmonella-like organisms.  Positive colonies were indicated by a bright pink 
coloration of the media or individual colonies.  If positive colonies were detected, they 
were transferred by the stab and streak method to Triple Sugar Iron agar (TSI; Neogen 
Corp.) and Lysine Iron agar (LIA; Neogen Corp.) for biochemical confirmation.  A 
positive biochemical test was indicated by a red slant and yellow to yellow-black butt 
(bottom portion of the tube) for TSI and a purple slant and purple to purple-black butt for 
LIA.  Positive samples on both TSI and LIA were then transferred to cryovials,
 
with the 
 60
addition of 1 mL of Tryptic Soy Broth (TSB; Neogen Corp.) with 30% sterile glycerol 
for future analysis.   
Upon completion of the 12-mo study, cryovialed samples were tested for 
Salmonella confirmation using latex agglutination.  Samples were removed from the 
freezer and allowed to thaw at room temperature with 0.5 ml being extracted and placed 
into 9.5 ml TSB as a growth medium.  After a 24 h incubation at 37?C, 1ml of sample 
was placed into 9 ml of TSB for another growth cycle.  After an additional 24 h 
inclubation was complete, 0.1 ml of sample was transferred to PCA and stored at 37?C 
for 24 h.  Individual colonies from the PCA were streaked for isolation onto BGS agar to 
ensure that the sample was positive for Salmonella-like species and that no contamination 
had occurred during storage, and incubated for 24 h at 37?C.  Samples were then tested 
using Salmonella O antisera (BD Difco, Rockville, MD) and AntiserumVi groups Band 
D (BD Difco, Rockville, MD). Individual colonies from the non-selective media were 
placed onto disposable agglutination sheets, with the addition of the antisera.  Both 
positive and negative controls were used to verify each sample.  A clinical rotator (Fisher 
Scientific, Pittsburg, PA) was used to aid in the agglutination process, as the samples 
must be gently swirled for 1-2 min.  Positive agglutinations were confirmed by the 
observance of coagulated areas within the sample.  Negative agglutinations showed no 
signs of coagulation and the sample retained its initial integrity. 
Statistical Analysis 
The experimental design of this study was a 2 X 12 factorial arrangement of a 
completely randomized design, representing the two hen strains (Hy-Line W-98 and 
Bovans White) and the 12 mo study period.  Plate counts were converted to base-10 
 61
logarithm values and subjected analysis of variance using PROC GLM and Fisher 
Protected LSD in SAS (SAS Institute, Cary, NC).  Frequencies were obtained using the 
PROC FREQ of SAS.  The values in Figure 1 represent the average of the 2 strains and 
replicates for a given period.  Results indicating a zero count in bacteria were set at the 
lowest detectable limit of 10 cfu / mL, while results indicating a too numerous to count 
(TNTC) measurement were set to a limit that was 1 log unit higher than the highest 
detectable plate count (100,000 cfu / mL).   
RESULTS AND DISCUSSION 
As shown in Figure 1, hen age significantly (P < 0.05) affected the microbial 
loads on each of the egg components tested, however hen strain did not have a significant 
(P > 0.05) impact on bacterial counts.  Interior shell APC counts were erratic (P < 0.05) 
and increased as much as 2 log units at points during the 12-mo period.  This is evident 
by the decrease in interior shell counts in period 2, coupled with their highest peaks in 
periods 8, 10, and 12.  Content counts remained minimal throughout the study period 
with only a slight increase in periods 5, 6, and 12.  This may be attributed to a condition 
of Osteomalacia found in the laying flock during period 4, which may explain the slight 
increase in the exterior shell counts and some of the variability early in the experiment.  
However, the birds were treated and egg production returned to normal by period 5.   
The frequency of Salmonella-positive samples, shown in Table 1, were low and 
are accounted for by a positive sample from the exterior shell in period 1, and a positive 
sample from the interior shell in period 6.  Contents showed no positive Salmonella 
samples throughout the course of the 12 mo study.  Each of these positive samples were 
determined to be so by  testing on Salmonella-selective BGS agar and TSI and LIA 
 62
biochemical tests, followed by  latex agglutination.  The incidence of Salmonella-
positives in either the interior shell or the exterior shell was 0.83% for 120 pooled sets. 
Both Salmonella-positive samples occurred in the Hy-Line W-98 layer strain. 
Based on these findings, one can conclude that there is a relationship between the 
mechanical properties of the inner shell membrane and the contents of the shell egg being 
free of pathogens.  Since no positive samples were discovered for the egg contents 
throughout the 12-mo study, it appears as though the shell?s physical barriers and 
complex network of shell membrane fibers are effective against bacteria entering the 
contents.  In essence, the interior membranes are able to trap the microorganisms before 
they reach the nutrient dense yolk.  It is also possible that certain natural antimicrobials, 
such as lysozyme also play a key role in reducing the number of bacteria found in the 
albumen.   One could also infer that the hens in the flock were negative for Salmonella 
colonization, since there was no transovarian deposition of Salmonella into the contents.   
However, Salmonella samples were found on both the exterior and interior 
portions of the shell egg.  The positive sample obtained on the exterior shell could 
possibly be explained by the egg coming into contact with contaminated material in the 
external environment.  It is not uncommon for the bacterial colonies found in fecal 
material to adhere to the exterior portion of the shell and survive there.  It is also possible 
that this could account for the positive sample that was obtained in the interior shell.  One 
theory is that the yolk and albumen contract shortly after lay, causing microorganisms to 
be pulled through the pores of the shell (Board, 1966).  If this is correct, one can assume 
that bacteria on the exterior shell could have theoretically moved into the inner shell 
 63
membranes and matrix.  Also, since Salmonella bacteria are equipped with flagella, they 
have the ability to move to areas which are more conducive to survival.   
Although it has been proven that as hen age increases, shell strength may become 
compromised, we did not find this to contribute to Salmonella contamination in our 
study.  The positive samples occurred in periods 1 and 6, while no positive samples were 
found toward the end of the research.   
Conclusion 
 In summary, the findings of this study suggest that environmental contamination 
in this facility may be more prevalent than colonization in the hens.  More research is 
needed to determine whether the shell membranes or the antimicrobials in the albumen 
play the primary role in protecting the egg contents from environmentally transferred 
microbial contamination. 
ACKNOWLEDGEMENTS 
 The authors would like to thank the North Carolina State University Piedmont 
Research Station staff for rearing the laying hens and helping to collect the eggs utilized 
in this study, the Auburn University Egg Quality Lab for their support and assistance, and 
the Poultry Products Safety and Quality Peaks of Excellence Program for funding this 
research. 
 64
Table 1. Frequency of Salmonella positives by strain, hen age, and sampling location             
  W- 98    
Bovans 
White  
Hen 
Age Exterior Shell 
Interior 
Shell 
Egg 
Contents  
Exterior 
Shell 
Interior 
Shell 
Egg 
Contents 
20 wk  1/5 0/5 0/5  0/5 0/5 0/5 
24 0/5 0/5 0/5 0/5 0/5 0/5 
28 0/5 0/5 0/5  0/5 0/5 0/5 
32 0/5 0/5 0/5 0/5 0/5 0/5 
36 0/5 0/5 0/5  0/5 0/5 0/5 
40 0/5  1/5 0/5 0/5 0/5 0/5 
44 0/5 0/5 0/5  0/5 0/5 0/5 
48 0/5 0/5 0/5 0/5 0/5 0/5 
52 0/5 0/5 0/5  0/5 0/5 0/5 
56 0/5 0/5 0/5 0/5 0/5 0/5 
60 0/5 0/5 0/5  0/5 0/5 0/5 
64 0/5 0/5 0/5 0/5 0/5 0/5 
 65
Figure 1.  Aerobic plate counts of the exterior shell, interior shell, and contents. 
 
 
APC Counts
0
1
2
3
4
5
12345678910112
Period
Lo
g 1
0
 c
f
u
/
m
l
Exterior
Interior 
Contents
 66
MANUSCRIPT II 
 
INFLUENCE OF HEN AGE AND THREE MOLTING TREATMENTS ON SHELL 
EGG EXTERIOR, INTERIOR, AND CONTENTS MICROFLORA AND 
SALMONELLA PREVALENCE DURING A SECOND PRODUCTION CYCLE 
 
ABSTRACT  
 Salmonellosis  is one of the most frequently reported foodborne illnesses in 
the world, and Salmonella Enteritidis (SE) is the serotype most associated with eggs.  
The objective of this study was to determine if increasing hen age and three different 
molting treatments influenced the total microflora counts or the prevalence of 
Salmonella spp. on the exterior of the egg shell, within the interior shell, or in the 
contents.  Eggs from Hy-Line W-98 and Bovans White layer strains were sampled 
approximately every 28 d from 70 to 114 wk of age, with the molting period from 66 
to 70 wk of age.  Layers were utilized from the 35
th
 North Carolina Layer 
Performance and Management Test (NCLP&MT), and managed under identical 
husbandry practices.  This study consisted of non-fasted, non-molted, and feed 
restricted treatments with the use of 135 eggs per layer strain, for a total of 270 eggs 
sampled per period.  The exterior, interior shell, and contents were spiral plated onto 
Plate Count agar (PCA) to calculate the total aerobic counts.  Additional pre-
enrichment, enrichment, conformational, and biochemical procedures were performed 
to test for the presence of Salmonella spp.  Hen age and molting treatment 
significantly (P<0.05) affected the microbial loads on all three egg components.
 67
Exterior, interior, yolk, and albumen counts increased during the molt period to as 
much as 1 log unit higher than the highest countable plate, which was 10
5.  
Exterior, 
interior, and contents counts rose (P<0.05) during period 15, with an increase 
(P<0.05) in the interior also in period 14, and in the contents in periods 14 and 17.  
There were a total of 360 egg pools and of those, 4 were positive Salmonella samples.  
Both the interior and exterior shell components and 2 of the 3 molting treatments had 
positive samples.  Of these positives, 4 were confirmed as Salmonella braenderup.  
Three positives were associated with the interior component, while 1 positive was 
associated with the exterior shell component. Three of the 4 samples were related to 
the non-fasted treatment, while the remaining positive was found in the non-molted 
treatment. 
INTRODUCTION 
 The relationship between the use of different molting procedures in laying hen 
flocks and Salmonella Enterica serovar enteritidis (SE) has been a topic of debate for 
many years.  Although molting is an important economic tool for the egg industry, 
some may argue that it places undue stress on the flock, suppressing the immune 
system and creating an environment suitable for SE growth.  In 2003, Holt reported 
that in a simulated flock situation, molted hens excreted higher SE numbers in feces, 
had increased counts in the internal organs, and were observed as having more 
intestinal inflammation.  He also discovered that the hens in this study were 100- to 
1000-fold more susceptible to SE organisms, making them potential vectors for 
transmission to uninfected birds in neighboring cages.  In a study conducted by 
Butcher and Miles (1994), it was determined that no matter what factors are involved 
 68
in causing increased susceptibility to SE infection, laying hens subjected to a forced 
molt by feed removal are being stressed and are more likely to shed Salmonella 
bacteria  as compared to non-molted hens. 
  Molting is a natural physiological process that occurs in chickens, fowl, and 
other avian species, which produces a cessation of egg production and permits the 
reproductive tract and plumage time to regenerate.  According to Park et al (2004), 
this gives hens time to recuperate from a long laying cycle while causing a state of 
reproductive quiescence, leading to a second cycle of eggs.   In 1994, Butcher and 
Miles reported that with proper procedures, it is possible to achieve a productive life 
from 70 to 105 wk of age and for an additional 25 to 30 wk if a second molt is used.  
This is important because laying hens are bred specifically for egg production and the 
intent of the egg industry is to make a profit.  However, although increased egg 
production is a primary focus, the welfare of the birds is also of great importance 
(Anderson and Koelkebeck, 2007).   
 There are several methods used to induce birds into a molt.  During a 
conventional induced molt, feed is removed or significantly limited, thereby inducing 
molt.  Although there has been debate as to which method of induced molting will 
attain the greatest production rate and egg quality, the traditional induced molt 
procedure is still the comparative basis used, however, many poultry companies, to 
meet animal welfare guidelines, are now using non-fasting programs.   It has also 
been reported that the removal of specific nutrients such as sodium (Whitehead and 
Shannon, 1974), iodine (Arrington et al, 1967), calcium (Gilbert and Blair, 1975), or 
zinc (Creger and Scott, 1977) will induce hens into a molt.  In 1962, Bell and Siller 
 69
conducted an experiment which revealed that hens fed a calcium deficient diet of less 
than 0.3% would cease egg production within 7-10 d.  Approaches that do not use 
complete feed withdrawal are becoming more desirable.  According to Biggs et al, 
2003, non-feed removal techniques are not less effective in recovering post-molt egg 
production.  These methods may include feeding diets high in corn gluten, wheat 
middlings, or a combination of corn and wheat.  These results appear to suggest that 
alternative molting methods may be more animal welfare friendly than the accepted 
feed withdrawal regimens.  However, Berry (2003) reported that these 
unconventional practices have not proven to be as effective in ceasing egg production 
and could potentially cause a decrease in lay without allowing a full rest period.  This 
could mean that the flock would not reach its full post-molt production potential once 
the molt has concluded.  Although it has been noted that an induced molting regimen 
shows higher incidences of SE (Holt and Porter, 1992), it is important to mention that 
many experiments used to study the nature of SE in laying flocks use inoculated hens 
as their test subjects.  Anderson et al (2001) questioned that because of the fact that it 
was difficult to determine if the results obtained in such studies were genuine 
reflections of the industry, or simply due to high levels of inoculum or commercial 
settings and breeds.   
MATERIALS AND METHODS 
 Eggs from two commercial layer strains, Hy-Line W-98 (HL) and Bovans 
White (BW) were used as the source for monitoring the microbial population on an in 
shell eggs.  The hens were managed under the guidelines outlined in the 35
th
 North 
Carolina Layer Performance and Management Test (NCLP&MT) (Anderson, 2005), 
 70
under identical husbandry practices.  Each period, on day 28, the previous 24 h of egg 
production was collected from five specific replicates of the utilized hen strains, by 
staff wearing laboratory gloves, hairnets, and jumpsuits, to eliminate potential human 
contamination, then placed in sterile plastic flats, packed in clean plastic bags, and 
positioned into a 30 dozen case.  The eggs were stored in a 4?C walk-in cooler until 
they were removed from the Piedmont, North Carolina facility by an Auburn 
University technician on the day of collection, and transported to the Auburn 
University Poultry Science Building where they were stored overnight in a 4?C walk-
in cooler before undergoing microbiological analyses the following day.  
 The eggs for this experiment were collected from five replicates from the HL 
and BW strains. A minimum of twelve eggs were collected from each of the 5 
replicates-strain combinations.  In order to ensure the minimum number of sound 
uncracked eggs, more eggs were collected than needed for analysis to compensate for 
any eggs that may have become cracked during transport to the laboratory.  This was 
accomplished by hand candling to ensure all shells were intact upon arrival.  During 
the course of this 12-mo experiment, three molting treatments, consisting of non-
fasted (NF), non-molted (NM control), and feed restricted (FR) groups were utilized 
as described by Anderson and Havenstein (2007).  Forty-five eggs from each 
replicate / strain / molt combination (135 eggs per respective strain) were randomized 
and the set of eggs was separated into groups of 9 egg pools for a total of 15 pooled 
sets per strain.  The microbiological procedures used for this study were obtained 
from the USDA Food Safety and Inspection Service Laboratory Guidebook (2004).  
Each individual egg was then placed into a small sterile rinsate bag with the addition 
 71
of 25 mL of Buffered Peptone Water (BPW; Neogen Corp., Lansing, MI).  The 
individual bags were then closed and shaken vigorously by hand for 1 min to ensure 
that the exterior shells were rinsed thoroughly.  Rinsates from individual eggs were 
then pooled into another sterile bag with a total of 9 rinsates per pool.  A small 
amount of the pooled rinsate was placed into sterile spiral plater cups and 50 ?l plated 
onto Plate Count agar (PCA; Neogen Corp.) using a WASP spiral plater 
(Microbiology International, Frederick, MD).  The additional rinsate was incubated at 
37?C for 24 h for further Salmonella testing.   
 The eggs were then removed from the original rinsate bag with sterile tongs 
and flamed by dipping in 70% ethanol and passing through a Bunsen burner.  With 
the exception of period 13 (molt period), eggs were cracked onto sterile foil and the 
contents (both yolk and albumen) were placed into a sterile stomacher bag, while the 
shells were placed into a sterile filter bag and gently crushed by hand.  However, 
during period 13 the contents were separated into yolk and albumen pools to 
determine if microbial counts differed between yolk and albumen and what possible 
Salmonella organisms could be found in the separated components as opposed to the 
contents as a whole.  The yolks were rolled on towels to remove any adhered albumen 
and each component was placed into a separate sterile bag.  The results indicated that 
there was no significant difference (P>0.05) between the microbial counts of the 
separated components, so yolk and albumen contents were analyzed together as 
whole egg for the remaining 11 periods.  Beginning in period 14 (one month post-
molt), the contents were placed in a stomacher bag and homogenized in a Seward? 
400 Circulator stomacher (Seward, Norfolk, UK) at 200 rpm for 1 min, and 10 mL of 
 72
contents was extracted and placed into another sterile stomacher bag with the addition 
of 90 mL of BPW to create a 1:10 dilution.  The 100 mL of contents / BPW was then 
homogenized in the stomacher for an additional 1 min at 200 rpm.  A small amount of 
the contents was placed into a sterile spiral plater cup to be spiral plated onto PCA.  
The remaining mixture was placed into an incubator at 37?C for 24 h for further 
Salmonella testing.   
 The filter bag containing the shells was gently crushed by hand and then 90 
mL of BPW and the shells were shaken by hand for 1 min to ensure proper washing 
of the interior shells and membranes.  A small amount of this rinsate was also placed 
into a sterile spiral plater cup for spiral plating onto PCA and the remaining rinsate 
was incubated for 24 h at 37?C for the first step in the enrichment process for 
Salmonella testing.  All spiral plated PCA plates from each of the three egg 
components (exterior rinse, interior rinse, and contents, with the addition of separate 
yolk and albumen plates from period 13) were incubated at 37?C for 24 h to 
determine total aerobic counts.   
 After a 24 h incubation period, each set of 9 pooled exterior and interior rinses 
or contents for each of the components was removed from the incubator and shaken 
by hand for 1 min to homogenize.  Then, 0.1 mL was removed from each bag and 
placed into 9.9 mL of Rappaport-Vassiliadis R10 (RV; Neogen Corp) enrichment 
broth.  Simultaneously, 0.5 mL was also removed from the incubated rinsate bag and 
placed into 9.5 mL of Tetrathionate-Hajna (TT; Neogen Corp.) enrichment broth.  
Both the RV and TT tubes were placed into a 42?C incubator for 24 h.  The 
remaining egg rinses and contents were then discarded.  The PCA plates that were 
 73
inoculated the previous day were removed from the incubator and read using a 
QCount? plate counter (Spiral Biotech, Inc., Norwood, MA), using the spiral plate 
function.   
 The RV and TT tubes were removed from the incubator after 24 h and 
vortexed to ensure homogeneity.  A 0.1 mL bioloop of inoculum was then removed 
from the tube and streaked for isolation onto Brilliant Green Sulfapyradine agar 
(BGS; Neogen Corp.) and Modified Lysine Iron agar (mLIA; Noegen Corp.).  The 
plates were then placed into a 37?C incubator for 24 h for the detection of Salmonella 
and Salmonella- like organisms.  Positive colonies were indicated by bright pink 
coloration of the media or colonies on BGS agar and purple to purple-black colonies 
on the mLIA agar.  If positive colonies were detected, they were transferred by the 
stab and streak method to Triple Sugar Iron agar (TSI; Neogen Corp.) and Lysine 
Iron agar (LIA; Neogen Corp.) for biochemical conformation.  A positive 
biochemical test was indicated by a red slant and yellow to yellow-black butt (bottom 
portion of the tube) for TSI and a purple slant and purple to purple-black butt for LIA.  
Samples that were positive on both TSI and LIA were then transferred to cryovials by 
removing colonies with a sterile loop, which contained 1 mL of Tryptic Soy Broth 
(TSB; Neogen Corp.), with 30% sterile glycerol for future analysis. 
 Upon completion of the 12 month study, cryovialed samples were tested for 
Salmonella conformation using latex agglutination.  Samples were removed from the 
freezer and allowed to thaw at room temperature with 0.5 mL being extracted and 
placed into 9.5 mL TSB.  After 24 h incubation at 37?C, 1 mL of sample was placed 
into 9 mL of TSB for another growth cycle.  After an additional 24 h incubation was 
 74
complete, 0.1 mL bioloop of sample was transferred to PCA, streaked for isolation, 
and incubated at 37?C for 24 h.  Individual colonies from PCA were selected and then 
streaked for isolation onto BGS agar and incubated for 24 h at 37?C, to ensure that 
the sample was positive for Salmonella-like species and that no contamination had 
occurred during storage, and incubated for 24 h at 37?C.  Samples were then tested 
using Salmonella O antisera (BD Difco, Rockville, MD) and Antiserum Vi groups B 
and D (BD Difco).  These two groups were chosen because they encompassed the 
Salmonella serotypes most commonly associated with eggs.  Individual colonies from 
the non-selective PCA media were placed onto disposable agglutination test sheets, 
with the addition of the antisera.  Both positive and negative controls were used to 
verify each sample.  A clinical rotator (Fisher Scientific, Pittsburg, PA) was utilized 
to aid in the agglutination process, as the samples were gently swirled for 1-2 min.  
Positive agglutinations were confirmed by the observance of coagulated areas within 
the sample.  Negative agglutinations showed no signs of coagulation and the sample 
retained its initial integrity.  Samples that were agglutination positive for Salmonella 
were then sent to the National Veterinary Services Laboratories (NVSL, Ames, IA) 
for serotyping. 
Statistical Analysis   
 The experimental design of the study was a 2 X 3 X 11 factorial arrangement 
of a completely randomized design, representing the two hen strains (HL and BW), 
the three molting treatments (NF, NM, and FR), and the 11 month study period in 
which the separated yolk and albumen components from the molt period were 
combined and analyzed as whole egg.  For the molt period (period 13), microbial 
 75
counts were analyzed as exterior shell, interior shell, yolk, and albumen using both 
the PROC GLM and PROC FREQ in SAS version 9.0 (SAS Institute, Cary, NC).  
Plate counts were converted to base-10 logarithm values and subjected to analysis of 
variance using PROC GLM and Fisher Protected LSD in SAS version 9.0.   
Frequencies were obtained using the PROC FREQ procedure of SAS.  Results 
indicating a zero count in bacteria were analyzed at the lowest detectable limit of 0.9 
cfu / mL, while results indicating a too numerous to count (TNTC) measurement were 
set to a limit that was 1 log unit higher than the highest detectable plate count 
(100,000 cfu / mL). 
RESULTS AND DISCUSSION 
 The values reported in figures 1-4 represent the average of the 2 strains, the 
three molting treatments, and replicates for a given period.  As shown in Figure 1, 
exterior, interior, yolk, and albumen counts, when compared to exterior, interior, and 
contents counts shown in Figure 2, increased during the molt period by as much as 1 
log unit higher than the highest countable plate, which exhibited a 10
5
 value.  In 
essence, the average interior counts for periods 14-24 indicate a 2 log decrease from 
those found during the molting period, while contents counts showed an approximate 
decrease of 3 logs, and exterior counts decreased by 2 logs.  This indicates that 
bacterial shedding during the molt period was higher than all other periods.  This is 
consistent with studies that suggest molting increases the frequency of bacterial 
shedding.  Hen age significantly (P<0.05) affected the microbial loads on all three 
tested egg components (Figure 3), while molting treatment had a significant (P<0.05) 
effect on the frequency of Salmonella positive samples (Figure 4).   
 76
  As shown in Figure 3, exterior, interior, and contents counts rose (P<0.05) 
during period 15, with an increase (P<0.05) in the interior counts in period 14, and in 
the contents in periods 14 and 17.  The increase in counts during the first two periods 
post-molt were potentially due to stress caused by the molt, while the increase in 
counts in later periods could be related to hen age and the resumption of egg 
production post-molt.  Although there was a significant difference (P<0.05) across 
periods, there were no differences (P>0.05) between populations of aerobic bacteria 
found within each molting treatment.  This observation suggests that molted and non-
molted birds shed the same amount of bacteria.  This contradicts that theory that non-
molted birds will shed fewer bacteria than non-fasted or feed-restricted birds. 
 However, the molting treatment, did have a significant (P<0.05) impact on the 
frequency of Salmonella positive samples.  The results, shown in Table 1, indicated 
that the greatest number of Salmonella positive samples occurred in the non-fasted 
and non-molted treatments.  This discovery contradicts the findings of pervious 
studies, which state that induced molting, including a feed restriction treatment, has 
been shown to increase shedding of Salmonella bacteria.  However, this experiment 
showed that the feed-restricted treatment had the lowest frequency of Salmonella, 
when compared to the other two treatments.  The discrepancy in results from previous 
studies could be due to the fact that this trial utilized the naturally occurring 
contamination and did not inoculate the hens in the beginning.  We were simply 
looking for the natural occurrence of SE with no predisposed bias.  Many studies 
similar to this one used hens infected with SE to ensure concentration in the gut, and 
therefore promote bacterial shedding.  The results obtained in Figure 5 also indicate 
 77
that an increase in hen age had an effect on Salmonella frequency.  It was discovered 
that the greatest occurrence was detected in period 21 when 3 interior and 1 exterior 
proved to be Salmonella positive.   
 All eggs produced by the two laying strains (BW and HL) during the 
collection period were utilized in this experiment. There were a total of 360 egg pools 
and of those, 4 were positive Salmonella samples.  Two components and 2 molting 
treatments had positive samples.  The four Salmonella braenderup samples were 
identified in period 21.  Three positives were associated with the interior component, 
while 1 positive was associated with the exterior shell component.  When comparing 
these results to the molting treatments used, it was revealed that 3 of the 4 positive 
samples were related to the NF treatment, while the remaining positive was found in 
the NM treatment. None of the Salmonella found in association with the shell eggs 
used for this study could be correlated to the samples taken of the environment.  
Although Salmonella infantis and ohio were discovered in the environment, these 
serotypes were not discovered in the eggs.  This would suggest that the serotypes 
indicated with the shell eggs were not due to environmental contamination, but could 
be due to fecal contamination from the hens.   
Conclusion 
 In summary, the findings of this study suggest that an induced feed-restricted 
molting treatment does not increase the incidence of Salmonella spp., including SE 
which has been documented to increase during molting.  Of the Salmonella positives 
discovered in this trial, none were found to be SE.  This is potentially due to the fact 
this experiment focused on the natural occurrence of SE, and no hens were inoculated 
 78
with bacteria in this study.  The results of specific serotyping indicated that the strains 
found in the shell eggs did not match those of the environmental samples.  Although 3 
of the 4 Salmonella braenderup positives were linked to the interior shell, no 
Salmonella was found in the contents (yolk and albumen) of the eggs.  This supports 
the theory that Salmonella infection could have occurred at some point through the 
duration of the study.  This mode of transmission would provide a viable opportunity 
for organisms to enter into the shell eggs.  Although bacteria can travel through the 
pores and into the interior shell membranes and matrices, the albumen does not 
provide a suitable growth medium, being deficient in nutrients and laden with 
antimicrobials.   
ACKNOWLEDGEMENTS 
 The authors would like to thank the North Carolina Department of Agriculture 
and Consumer Services, Piedmont Research Station staff for rearing the laying hens 
and helping to collect the eggs utilized in this study, the Auburn University Egg 
Quality Lab for their support and assistance, and the Poultry Products Safety and 
Quality Peaks of Excellence Program for funding this research. 
 
 
 
 
 
 79
 
Figure 1. Aerobic bacteria plate counts for the molting period. 
 
 
Period 13 APC
0
2
4
6
8
10
Interior  Yolk  Albumen  Exterior
Component Tested
Lo
g 1
0
 c
f
u/m
l
FR
NF
NM
 80
Figure 2. Aerobic bacteria plate counts for molting treatments from periods 14-24. 
 
Molt Treatments Periods 14-24
0
1
2
3
4
5
6
Interior  Contents  Exterior
Components Tested
L
og 1
0
 
c
f
u/
m
l
FR
NF
NM
 81
Figure 3. Aerobic bacteria counts shown per periods 14-24. 
 APC Counts
0
1
2
3
4
5
6
14 15 16 17 18 19 20 21 22 23 24
Period
L
o
g
 10 cf
u
/
m
l
Interior
Contents
Ex ter ior
 82
Figure 4. The frequency of Salmonella positive samples per molting treatment. 
Salmonella Frequency Per Molting Treatment
0
0.5
1
1.5
2
2.5
3
3.5
NF FR NM
Molt Treatment
N
u
m
b
er
 o
f
 
P
o
sit
i
ves 
P
e
r
 T
r
eat
m
e
n
t
 83
Figure 5. The frequency of Salmonella suspect positive samples by period. 
 
 
Salmonella Frequency
0
0.5
1
1.5
2
2.5
3
3.5
13 14 15 16 17 18 19 20 21 22 23 24
Period
S
a
l
m
o
n
e
l
l
a
 P
o
si
t
i
ves P
e
r
 
360 P
o
o
l
ed
 S
e
t
s
Exterior
Interior
 84
Table 1.  Positive samples indicated by serotyping from the National Veterinary 
Services Laboratory. 
Period Location Molting 
Treatment 
Serotype 
21 Exterior NF S. braenderup 
21 Interior NF S. braenderup 
21 Interior NF S. braenderup 
21 Interior NF S. braenderup 
 85
MANUSCRIPT III 
 
EFFECTS OF CITRIC ACID AND PASTEURIZATION ON THE FUNCTIONAL 
AND CHEMICAL ANALYSES OF LIQUID WHOLE EGG 
 
ABSTRACT  
Although a large portion of the eggs produced today are sold in the form of shelled 
eggs, approximately 30 percent of the eggs produced by the industry are broken out 
and marketed as pasteurized liquid egg products.  The purpose of this research was to 
determine if the addition of citric acid to pasteurized whole egg, as well as the 
pasteurization process itself, influenced the results of functional experiments and 
chemical analyses.  Liquid whole egg was obtained from egg processing facilities 
which supplemented a portion of its pasteurized product with citric acid.  Four 
treatments consisting of  unpasteurized, pasteurized, unpasteurized with citric acid, 
and pasteurized with citric acid liquid whole egg were extracted in 5 lb sample 
quantities.  Samples were taken during three separate processing periods to ensure 
that egg lots were not duplicated.  Each treatment was analyzed for pH, total 
moisture, total fat, total protein, sponge cake volume, sponge cake texture profile 
analysis (TPA), custard weep, and custard gel bloom.  The results of this study show 
that neither pasteurization nor citric acid supplementation had a true, practical impact 
on the chemical composition of whole egg products.  The use of the CEM provides 
rapid, accurate results for total moisture, fat, and protein of whole egg samples.  
Although further research is needed to achieve AOAC 
 86
approval, the results of this study indicate that this is a possible alternative to traditional 
proximate analysis procedures used for egg products. 
 The addition of citric acid to whole egg products created a more voluminous cake 
than those that were left un-supplemented. The theory that pasteurization is detrimental to 
whole egg functionality was not evident in this study.  Both treatments that utilized the 
pasteurization process performed best in both custard weep and bloom, with the PC 
treatment also having a high sponge cake volume. 
INTRODUCTION 
Whole Egg Pasteurization 
 Although a large portion of the eggs produced today are sold in the form of 
shelled eggs, approximately 30 percent of the eggs produced by the industry are broken 
out and marketed as pasteurized liquid whole egg products.  According to the FSIS 
(2007) approximately 30% of the 76 billion eggs consumed in 2007 were in the form of 
egg products, or eggs that have been removed from the shell and pasteurized.  From the 
baking industry to specialty products, pasteurized products are developed utilizing whole 
egg mixtures, as well as separated yolk and albumen components.  Many consumers 
favor these products because of the idea that pasteurization aids in the destruction of 
harmful bacteria and because they are perceived as ?safer? than shelled eggs.    
 It is extremely important to maintain the functional properties of liquid whole egg 
because it is so commonly used as an ingredient in foods products.  Studies such as those 
done by Baldwin (1986) have described how the application of heat can both positively 
and negatively impact the functionality of liquid whole egg.  The challenge for the 
industry is striking a balance between ensuring consumer safety and maintaining the 
 87
functional integrity of the product.  Because of this, Dawson and Martinez-Dawson 
(1998) conducted a study that evaluated what effect specific pasteurization 
times/temperatures had on the functionality of liquid whole egg products.  By using 
Response Surface Analysis (RSA), they were able to show the effects of a range of 
different pasteurization time-temperature combinations on quality and functional factors 
of liquid egg.  They discovered that when measuring cake height volume, there was more 
of time dependence than temperature dependence.  This is possibly due to the fact that 
when eggs are held at high temperatures for long periods of time, their proteins begin to 
denature and unfold, making them incapable of trapping the large amounts of air needed 
to create a greater cake volume.  Solids content followed this same trend, being more 
time dependent.  
 Many times, citric acid is added to pasteurized whole egg products.  There is little 
research discussing the reasoning behind this addition and its effect on cooked products 
in comparison to whole egg without the added citric acid.  However, there is an obvious 
decrease in pH because of the acidic characteristics and one could conclude that this 
addition combined with pasteurization, could potentially have a positive impact on 
bacterial elimination.  By definition, citric acid is a weak organic acid that is mostly 
associated with citrus fruits.  It is a natural preservative and is often found in candies and 
soft drinks that require a sour flavor.  Though it is widely used in the egg industry, there 
is not much research on the impact of this additive on the functionality of the liquid 
whole eggs.  
 
 
 88
Whole Egg Functionality 
 Although eggs provide many functional applications, one of the most common is 
the ability of eggs to coagulate or form a solid when heated (Stadelman, 1999).  Unlike 
carbohydrate gels, which solidify upon cooling, protein gels such as the ones found in 
egg custard, exhibit the gelation process upon heating.  In basic terms, a gel is an 
intermediate phase between a solid and a liquid.  However, technically, it is defined as a 
substantially diluted system which exhibits no steady state flow (Ferry, 1961).  During 
the process of whole egg gelation, proteins are first denatured into an intermediate gel 
state, in which the proteins are unfolded to expose functional groups.  This progression 
allows for the formation of the protein bonding network that eventually forms the solid 
state gel.  Protein gels are irreversible, meaning that upon either re-heating or cooling, 
they will not return to their former liquid state.  This is in contrast to gelatin gels, such as 
those seen in Jello? products, in which applying heat to the solid form will cause bond 
breakage, resulting in a return to the liquid state.  
 Another functional property of whole eggs is their ability to be used as both a 
leavening and humectant agent in bakery products.  Researchers often use sponge and 
angel food cakes to study the functionality of whole egg and albumen.  Although more 
research is needed to determine the functional effects of whole eggs in food products, 
various experiments have utilized sponge cake volume to determine leavening aspects.  
In a study conducted by Jones et al (1999), it was determined that increasing hen age 
demonstrated an increase in sponge cake volume.  In theory, the more volume a cake 
presents represents a better quality egg.  Although this finding is in contrast to the 
hypothesis that eggs of older hens are lower in quality than those of younger birds, the 
 89
effect on functionality could be due to the change in egg composition in correlation with 
increasing hen age.   
MATERIALS AND METHODS 
 Four treatments of liquid whole egg, pasteurized (P), unpasteurized (R) (control), 
pasteurized with citric acid (PC), and unpasteurized with citric acid (RC) (control) were 
sampled from egg processing facilities during three non-consecutive processing periods.  
Eggs were collected on the day of process in sterile plastic bags with secure screw-top 
closures to prevent leakage.  Samples were gathered by facility staff wearing sterile 
gloves, hairnets, and jumpsuits to prevent human contamination, and assembled into 5 lb 
quantities with three replicates (three 5 lb bags) representing each of the four treatments. 
The egg products were then shipped from the facility via overnight shipping in boxed 
coolers containing frozen cooler packs.  They arrived at the Auburn University Poultry 
Science Building by 10:00 a.m. 1 day post process, and chemical analyses began 
immediately.   
 Beginning 1 day post process, the liquid egg products were measured for pH 
using an Accumet? excel XL 20 pH/Conductivity meter (Fisher Scientific, Pittsburg, 
PA).  Total moisture and fat were measured using the CEM Microwave Technology 
Smart Trac? system in conjunction with the CEM Microwave Phoenix? system (CEM 
Corp., Matthews, NC).  Protein content was calculated based on the results of CEM  
Smart Trac? and Phoenix? data.  
 
 
 
 90
Chemical Analysis 
pH Measurement 
 Upon arrival at Auburn University, samples were tested for pH.  This procedure 
was done with the use of an Accumet? excel XL 20 pH/Conductivity meter.  Once 
calibrated, a small amount of egg sample was placed into a 50 mL beaker and the probe 
of the meter placed into the sample.   
CEM Microwave Technology 
 Once whole egg samples had been tested using AOAC analytical methods, those 
results were used to calibrate the CEM Microwave Technology equipment.  To test the 
total moisture content of the eggs, specially designed CEM sample pads were placed onto 
the internal balance using tongs, and the balance was tared and 2.0-2.5 grams of egg 
sample placed onto the pads.  After the balance reached a stable weight, a reading was 
given as to the exact weight of the sample in units carried out to four decimal places.  
Simultaneously, the internal drying oven began to dry the weighed sample at 100?C until 
all moisture was removed.  This process was 10 min in length.  Once dry, the total 
moisture percentage reading appeared on the CEM results screen and total fat analysis 
could begin.  Samples were removed from the internal balance with tongs and placed 
onto the CEM preparation station where they were gently rolled in a thin layer of CEM 
wrap.  The rolled sample was then placed directly into the fat analysis tube and 
compressed to approximately .75 in before being placed into the Smart Trac? system.  
After the analysis tube was placed into the system, it provided a direct measurement of 
total fat, including both free and chemically bound fat in solids, liquids and slurries.
 To test for the protein content of egg samples using the CEM, it was imperative to 
 91
employ the use of the CEM Phoenix Muffel Furnace?.  To complete this analysis,  
disposable quartz ashing crucibles containing two small circular pads were placed onto an 
analytical balance using tongs.  The disposable crucibles and pads were weighed and 
tared and 5.0-6.0 grams of sample placed inside.  They were then removed from the 
balance with tongs and placed into the Phoenix Muffle Furnace? for ashing at 550?C for 
35 min.  Once ashing had occurred, the samples were removed and quickly reweighed to 
obtain the amount of ash.  Once these three measurements (moisture, fat, and ash) were 
obtained, it was possible to calculate the percentage of protein in the samples using the 
formula: 
100% - % total moisture - % total fat ? % ash = % protein 
Functional Analysis 
Sponge Cake Volume Determination 
 To determine the volume of sponge cakes made with whole egg product, a 
Viking? (Viking Corp., Greenwood, MS) oven  was pre-heated to 350?F approximately 
20 min before the first cakes were added to it time to maintain a constant temperature.  
Using a 5 quart Kitchen Aid? (Kitchen Aid, Inc., St. Joseph, MI) mixing bowl, 138 
grams of whole egg was added along with 89.4 grams of granulated sugar.  The bowl was 
then placed onto a Kitchen Aid Artisan Series? (Kitchen Aid, Inc., St. Joseph, MI)  
mixer and a balloon whisk attachment attached.  Samples were then mixed for 15 sec at 
speed 2 and for an additional 4 min at speed 10.  Once complete, 2 mL of imitation 
vanilla flavoring was added to the mixture and homogenized for an additional 4 sec at 
speed 6.  Once mixed, 66 grams of cake flour was added to the batter in four equal 
portions and whisked gently with a hand whisk for approximately 20 strokes per flour 
 92
addition.  Cake pans with a pre-determined rapeseed volume were placed onto a balance 
and 75 grams of cake batter weighed into tared cake pans consisting of three sub-
replicates per replicate.  The cakes were then placed into the pre-heated ovens and baked 
for 17 min, removed after cooking and placed inverted onto cooling racks for at least 2 hr 
post bake.  After cooling, the cakes were turned upright and the tops lightly dusted with 
cake flour to ensure that the rapeseed did not stick to the cakes.  The remaining volume of 
the pan was filled with rapeseed and leveled off using a small metal spatula until the 
rapeseed was flush with top of the pan.  The pre-determined volume of the cake pan was 
then recorded, and the rapeseed was measured into a graduated cylinder using a funnel.  
The volume of the rapeseed was recorded and the cakes were saved for TPA analysis.    
The cake volume was determined using the following formula: 
Pre-determined pan volume ? rapeseed volume = cake volume. 
Sponge Cake TPA Analysis 
 The texture profile analysis of the sponge cake samples was done using a Stable 
Micro System?s TA.XT Plus Texture Analyzer (Texture Technologies Corp., Marietta, 
GA).  The TPA function of this equipment measures hardness, sponginess, gumminess, 
resilience, chewiness, cohesiveness, and force, graphically displaying the results onto the 
computer to which it is attached.  Once selecting the TPA function from the project list, 
cake samples were placed under a cylindrical Texture Analyzer attachment to ensure an 
even measurement.  The force at which the cakes will be compressed is then calibrated 
using a 5 kg load cell and a 2 kg weight.  Once completed, the cakes are then compressed 
twice, with the probe taking both the first and second peak measurements at 4 cm.   
 
 93
Custard Weep 
 Custards are a product of the gelation of a protein based product that gels upon 
heating and displays an irreversible process, unlike sugar based gels.  The egg custard 
samples were prepared by first pre-heating Viking ovens to 350?F.  In a mixing bowl, 
250 mL of whole egg, 0.5 c of sugar, 1.5 tsp vanilla, and 0.25 tsp salt was added.  The 
ingredients were then placed on the Kitchen Aid mixer and a balloon whisk attached.  
Custard samples were blended for 1 min at speed 4, and 3 c of hot whole milk added 
immediately following the 1 min blend.  The custard mixture was then measured to 100 
mL and poured into 4 oz ramekins that were lightly sprayed with cooking spray. 
Simultaneously, 60 grams of the custard mixture was placed into 100 mL beakers that 
would be used to measure the gel force.  Three sub-replicates of each replicate were taken 
to test for product variance.  The samples were then placed into a pan of hot water 
measuring 0.5 in from the top of the ramekins and placed into the ovens where they were 
baked for 40 min.  Once baked, a small metal spatula was inserted into the center of the 
custard to ensure that it was properly cooked.  Samples were then placed onto cooling 
racks and allowed to come to room temperature (approximately 1.5 hr.).  While the 
samples cooled, sample containers were prepared for weep measurement.  The lid of the 
plastic container was placed onto a balance.   Using gloves to prevent the addition of 
moisture and oils from human hands, 5 Whatman weighing papers (110 mm) were added 
to the lid to ensure that any residual moisture from the custards would be absorbed.  The 
containers were then labeled with the weight of the Whatman papers.  Once the custards 
had finished cooling, the ramekins were inverted to release the sample onto the weighing 
papers in the custard container.  The custards prepared in the beakers were covered with 
 94
Fisher Scientific Parafilm? wrap and both sets of custards were placed into a 4?C walk-
in cooler for 18-24 hr.   
 Once the samples were chilled for the appropriate time, the weep samples were 
removed from the cooler and gently removed from the Whatman paper.  The lid of the 
container, the weighing papers, and the residual weep was weighed on a balance and the 
weight recorded.  The amount of custard weep was then calculated using the formula:  
Final weight of lid, weep, and paper ? initial weight of lid and paper. 
Custard Bloom 
The custard samples prepared in the 100 mL beakers were removed from the 
cooler and taken to the lab for force analysis using the Texture Analyzer.  Using a 5 kg 
load cell and a 2 kg weight, the force of compression was calibrated.  As the custard was 
compressed 4 cm, the force results were displayed on the screen.  
Statistical Analysis 
The experimental design of this study was a Completely Randomized design 
(CRD).  Statistical analysis was done using the SAS? Statistical Package version 9.0 
(SAS Institute, Cary, NC).  Averages of the samples taken from the sub-replicates were 
analyzed using the General Linear Model (PROC GLM) in SAS and means found to be 
significantly different were analyzed using Fisher?s Protected LSD.   
RESULTS AND DISCUSSION 
 Although the treatments containing citric acid (PC and RC) were slightly lower in 
pH, this was not enough to create a statistical difference when compared to the non-citric 
treatments.  Egg processors are choosing to provide companies and consumers with a 
slightly more acidic product because those with a lower acidity have been shown to have 
 95
a longer shelf-life post-process.  Although the citric products perform as well as non-
citric chemically, there are issues concerning functionally, especially in bakery products, 
which needs to be addressed through further research. 
Once the pH measurements were taken, chemical analyses were performed on 
each of the treatments.  This was done using the CEM Microwave Technology Smart 
Trac? System, which performs both total moisture and total fat much more quickly than 
conventional methods.   There were no statistical differences (P>0.05)  in total moisture, 
fat, or protein between any of the treatments. 
  It was discovered that the treatments with citric acid supplementation were more 
effective in producing a high volume sponge cake and exhibited a positive impact during 
TPA analysis.  As shown in Figures 1 and 2, the treatment that contained citric acid was 
significantly different (P<0.05) from the P treatment.  This does not concur with the 
theory that citric acid can be detrimental to baking products.  Although the exact reason 
for this needs further research, it can be speculated that citric acid acts as a mechanical 
barrier which protects certain proteins from denaturation during the pasteurization 
process.  Although the P treatment was observed to be a softer cake, displaying lower 
force measurements, this is not necessarily a positive aspect.  The P treatment cakes were 
stickier upon visual observation and exhibited aspects of being gummy in texture and 
having a potentially chewy mouthfeel.  These observations were verified by the results of 
TPA analysis, indicating that the PC treatment possessed the qualities of a more ideal 
sponge cake.   
Although citric acid improved the functionality of whole egg products in a baking 
sense, it seemed that pasteurization had a greater impact on the functionality of egg based 
 96
gels.  The results of custard bloom and weep, shown in Figures 3 and 4, indicated that the 
two pasteurized treatments (P and PC, respectively) had a more positive impact on gel 
strength and formation. As the figures show, the P and PC treatments displayed a higher 
bloom force, meaning that the custard was more stable.  In contrast, the non-pasteurized 
treatments (RC and R) showed weaker gels with more weep.   
Conclusions 
The results of this study show that neither pasteurization nor citric acid 
supplementation had a true, practical impact on the chemical composition of whole egg 
products.    The results of the functional portion of this experiment revealed that the 
addition of citric acid to whole egg products created a more voluminous cake than those 
that were left un-supplemented.  It seems that the addition of certain chemical agents 
such as citric acid, provide a mechanical barrier that protect whole egg proteins from the 
damaging effects of pasteurization, in turn, protecting their functional properties. 
Although more research is needed to discover the chemical mechanisms that drive the 
reactions between egg proteins and citric acid, the results of this study conclude that 
supplementation with this product may improve the functionality of whole egg products.  
It was also discovered that the pasteurization process in general improved the stability of 
custards.  The results of analysis of the P and PC treatments indicated that the egg-based 
gels were stronger and more stable than those that did not utilize pasteurization.   
 
 
 
 
 97
Figure 1.  Sponge cake volume 
 
Sponge Cake Volume
0
100
200
300
400
PRPCRC
Treatment
Volum
e
 (
m
L)
 
 
P<0.05 
 
 
 
 
 
 
 
 
 
 
 
 98
Figure 2.  Sponge cake texture profile analysis for force (hardness), gumminess, and 
chewiness 
 
Sponge Cake TPA
0
200
400
600
800
1000
PRPCRC
Treatment
gr
a
m
s
Force Gumminess Chewiness
 
 
P<0.05 
 
 
 
 
 
 
 
 
 99
Figure 3.  Custard bloom as analyzed using the Texture Technologies Texture Analyzer 
 
Custard Bloom
0
250
500
750
1000
1250
1500
PPCRRC
Treatment
Force (g)
 
 
P<0.05 
 
 
 
 
 
 
 
 
 
 100
 
Figure 4.  Custard weep measurements 
 
Custard Weep
0
5
10
15
20
PPCRRC
Treatment
W
eep
 (
g
)
 
P<0.05
 101
MANUSCRIPT IV 
 
EFFECTS OF TRIETHYL CITRATE WHIPPING AGENT AND 
PASTEURIZATION ON THE FUNCTIONAL AND CHEMICAL ANALYSES OF 
LIQUID ALBUMEN 
 
ABSTRACT 
 Egg albumen plays many roles in the creation of bakery products.  It is used to 
make angel food cakes, meringues, candies, and many other confections.  Because of 
this, egg processors are marketing pasteurized egg albumen with added whipping 
agents.  However, there have been questions as to whether it performs as well as fresh 
albumen in functionality studies.  The purpose of this research was to determine if the 
addition of triethyl citrate whipping agent to pasteurized albumen, as well as the 
pasteurization process itself, influenced the results of functional experiments and 
chemical analyses.  Liquid albumen was obtained from egg processing facilities 
which supplemented a portion of its pasteurized product with triethyl citrate.  Four 
treatments consisting of  unpasteurized, pasteurized, unpasteurized with whipping 
agent, and pasteurized with whipping agent liquid albumen were extracted in 5 lb 
sample quantities.  Samples were taken during three separate processing periods.  
Each treatment was analyzed for pH, total moisture, total fat, total protein, angel food 
cake volume, angel food cake texture profile analysis (TPA), and whipping height.  
There were no statistical differences (P>0.05) observed for the pH measurements of 
each of the four treatments, as well as no differences (P>0.05) in total moisture, total
 102
 fat, or total protein.    While no differences (P>0.05) were found in whipping height 
measurements, there was a difference (P<0.05) observed in the angel food cake 
volume.  The pasteurized (P) treatment had the poorest performance overall, with the 
pasteurized with whipping agent (PW) showing the greatest angel food cake volume.  
These results show that the addition of triethyl citrate whipping agent has a positive 
impact on angel food cake volume.  The same results hold true for TPA analysis, 
where the PW treatment displayed the most positive and desirable characteristics. 
INTRODUCTION 
Albumen Pasteurization 
 It is no surprise that the pasteurization of various egg components provides a 
safer product, essentially free of microbial contamination, to consumers.  However, 
the positive aspects of pasteurization are counteracted with the negative impact it has 
on functionality, especially in the baking industry.  Pasteurized liquid egg whites are 
used in many pre-packaged products and are sold in liquid form in grocery stores.  
Often, consumers will buy such products and expect them to perform in the same 
manner as a shelled egg in their cooked foods.  However, this is not always the case.   
 One function of egg whites in food products is to produce foam.  Foam is 
created when air is trapped between the protein matrices when whipping is applied.  
In shelled eggs, it is important to make sure that the eggs used for this function are 
produced from young flocks.  As hen age increases, egg quality potentially decreases.  
This can cause the albumen component to become thin and watery, instead of thick 
and distinct, thus creating less voluminous foam.  This aspect can be detrimental to 
 103
the baking industry, with the fact being that eggs used from older flocks may produce 
cakes with less volume (Cunningham, et al 1960). 
 Water is the primary component of egg albumen (83%), while it also contains 
protein and only trace amounts of fats and sugars.  The main proteins found are 
ovalbumin (54%), conalbumin (13%), ovamucoid (11%), lysozyme (3.5%) which 
also serves as a natural antimicrobial, globulins (8%), and ovomucin (1.5%) (Tanner, 
2007).  The protein partially unravels and forms a good foaming agent. Foam is 
created when the protein forms a stable film around the included air. Studies show 
that the best foam is created when the protein is only partially unraveled. Over 
beating egg whites destabilizes the foam by fully unraveling the protein molecules. 
The protein is elastic, so when the egg white is cooked, and the air expands, the white 
stretches then sets in the expanded position.  Although albumen is comprised of 
several different proteins, only a few are responsible for foam formation and stability.  
According to Cotterill and Winter (1955) the globulins create the actual foam 
formation, while the ovomucin-lysozyme complex aids in foam stability.  Ovalbumin 
and conalbumin add heat-setting properties. 
 Both dry and liquid egg albumen may sometimes contain additives that aid in 
foaming capabilities.  A common additive is triethyl citrate, a form of citric acid.  
This is commonly used in pasteurized albumen products that have experienced some 
protein denaturation due to heat.  Another additive is sodium lauryl sulfate, which is 
added to improve foaming properties.  Triethyl citrate is used as a high boiling 
solvent and plasticizer for vinyl resins and cellulose acetates. It is a plasticizer 
permitted in the field of food additives, food contact material, medical, and 
 104
pharmaceutical products. Sodium lauryl sulfate is a detergent and surfactant found in 
many personal care products (toothpaste, shampoo, etc.). It is an inexpensive and very 
effective foamer.  These aids are capable of giving the product whipping properties 
that are comparable to, or even better than, the natural untreated product.  The type of 
additive used is dependent on the type of product being produced.  For instance, 
although no fully understood, triethyl citrate seems to be more preferable in liquid 
frozen or pasteurized whites, while sodium lauryl sulfate seems to work well with 
dried albumen. 
Albumen Functionality 
 Proteins are made of long chains of amino acids. The proteins in an egg white 
are globular proteins, which means that the long protein molecule is twisted and 
folded and curled up into a more or less spherical shape. A variety of weak chemical 
bonds keep the protein curled up tight as it drifts placidly in the water that surrounds 
it.  When you beat raw egg whites to make a souffl? or a meringue, you incorporate 
air bubbles into the water-protein solution. Adding air bubbles to egg whites unfolds 
those egg proteins just as certainly as heating them.   
 Some amino acids are attracted to water; they?re hydrophilic, or water-loving. 
Other amino acids are repelled by water; they are hydrophobic, or water-fearing.  
Egg-white proteins contain both hydrophilic and hydrophobic amino acids. When the 
protein is curled up, the hydrophobic amino acids are packed in the center away from 
the water and the hydrophilic ones are on the outside closer to the water. When an 
egg protein is up against an air bubble, part of that protein is exposed to air and part is 
still in water. The protein uncurls so that its hydrophilic parts can be immersed in the 
 105
water, and its hydrophobic parts can stick into the air. Once the proteins uncurl, they 
bond with each other, just as they did when heated, creating a network that can hold 
the air in place. 
 The foaming properties of albumen have long been praised for their role in 
confectionary functionality.  Often, egg white foams are associated with souffl?s, 
meringues, divinity, and angel food cakes. Because of its low fat content, angel food 
cake is frequently observed as being a healthier alternative to satisfying a person?s 
sweet tooth.  One of the most noticeable factors of this product is its light, airy 
texture, and its ability to produce a pleasantly sweet taste.  The lightness of texture is 
due to the trapping of air by the proteins in the process mentioned above, while the 
sweet taste is due to the addition of sugar into the batter.  However, this addition does 
more than just provide a delicious flavor.  If added properly, it will help stabilize the 
foam.  If sugar is added before foam formation occurs, it slows the denaturation of the 
proteins because it competes with the other molecules for access to water.  This does 
not allow the proteins to trap the air and create a stable foam.  However, if the sugar 
is added to the mixture after the foam is formed, it helps to generate stability, by 
adding a touch of viscosity between the trapped air bubbles.  If done properly, this 
should produce a stable foam with less weep. 
MATERIALS AND METHODS 
 
 Four treatments of liquid albumen consisting of pasteurized (PA), 
unpasteurized (RA), pasteurized with whipping agent (PW) and unpasteurized with 
whipping agent (RW) were obtained from egg processing facilities during three non-
consecutive processing periods.  Research samples were collected on-site at the 
 106
processing facility by employees wearing laboratory gloves, jumpsuits, and hairnets 
to prevent contamination, and divided into three 5 lb shipment bags per respective 
treatment.  The albumen samples were shipped overnight to the Auburn University 
Egg Quality and Safety Laboratory on the same day they were processed.  They 
arrived to Auburn 1 d post-process in tightly sealed shipment bags placed in sealed 
shipment coolers containing freezer packs.  The cooler containing the albumen was 
then placed into a labeled box for shipping.   
 Upon arrival, the shipments were opened and the bags of albumen separated 
into sets of 3 bags per treatment, for a total of 12 bags during each processing period.  
Each bag was considered to be a replicate, creating a total of 3 replicates per 
treatment per processing period, and a total of 9 replicates per treatment throughout 
the course of the study.  Each replicate (bag) was then further broken into 3 sub-
replicates to ensure that a large enough population of the replicate was sampled.  
Three samples were taken from each sub-replicate, for a total of 9 samples per 
replicate per processing period.  Throughout the study, a total of 324 albumen 
samples were analyzed for chemical composition, whipping height, and angel food 
cake volume.   
Chemical Analysis 
 Beginning 1 day post process the liquid albumen was measured for pH 
readings using an Accumet? excel XL 20 pH / Conductivity meter (Fisher Scientific, 
Pittsburg, PA).  The meter was first calibrated using solutions of a known pH.  A 
small amount of sample, approximately 50 mL was placed into a beaker and the 
probe of the pH meter inserted into the sample. Total moisture, fat, and protein were 
 107
measured using the CEM Microwave Technology Smart Trac? system in conjunction 
with the CEM Microwave Phoenix? system (CEM Corp., Matthews, NC). To test the 
total moisture content of the albumen, specially designed CEM sample pads were 
placed onto the internal balance using tongs.  Once placed, the balance was tared and 
2.0-2.5 grams of albumen placed onto the pads.  Once the balance was stabilized, a 
reading was given as to the exact weight of the sample in units carried out to four 
decimal places.  Simultaneously, the internal drying oven began to dry the weighed 
sample at 100?C until all moisture was removed.  This process was 5 min in length.  
Once dry, the total moisture percentage reading was transferred on the CEM to the 
results database and total fat analysis could begin.  Samples were removed from the 
internal balance with tongs and placed onto the CEM preparation station where they 
were gently rolled in a thin layer of CEM wrap.  The rolled sample was then placed 
directly into the fat analysis tube and compressed to approximately .75 in before 
being placed into the Smart Trac? system.  Once the analysis tube was placed into 
the system, it provided a direct measurement of total fat, including both free and 
chemically bound fat in solids, liquids and slurries. 
 To test for the protein content of albumen using the CEM, it was imperative to 
employ the use of the CEM Phoenix Muffel Furnace.  To complete this analysis, 
quartz ashing crucibles containing two small circular pads, were placed onto an 
analytical balance using tongs.  The quartz crucibles and pads were weighed and tared 
and 5.0-6.0 grams of albumen placed inside.  They were then removed from the 
balance with tongs and placed into the Phoenix Muffle Furnace? for ashing at 550?C 
for 35 min.  Once ashed, the samples were removed and quickly reweighed to obtain 
 108
the amount of ash.  Once these three measurements (moisture, fat, and ash) were 
obtained, it was possible to obtain the amount of protein in the samples, using the 
formula for percentage of protein: 
100% - % total moisture - % total fat ? % ash = % protein 
Functionality 
Angel Food Cake Volume 
 To determine the volume of angel food cakes made with albumen products, a 
Viking? (Viking Corp., Greenwood, MS) oven  was pre-heated to 350?F 
approximately 20 min before the first cakes were added, to give it time to maintain a 
constant temperature.  Using a 5 quart mixing bowl, 180 mL of albumen was added 
along with 2.70 grams of cream of tartar and 0.90 grams of NaCl.  The bowl was then 
placed onto a Kitchen Aid Artisan Series? (Kitchen Aid, Inc., St. Joseph, MI)   mixer 
and a balloon whisk attachment attached.  Samples were then mixed for 45 sec at 
speed 10.  Once complete, 2 mL of imitation vanilla flavoring was added to the 
mixture and homogenized for an additional 4 sec at speed 6.  After mixing, 66 grams 
of cake flour with the addition of 46 grams of granulated sugar was twice sifted, and 
added to the batter in 3 equal portions, gently folding it into the batter with a hand 
whisk for approximately 20 strokes per flour addition.  This process ensured that the 
foam which formed was not disrupted.  Cake pans with a pre-determined rapeseed 
volume were placed onto a balance and 75 grams of batter weighed into the tared 
pans, in a ratio of three samples per sub-replicate.  The cakes were then placed into 
the pre-heated ovens and baked for 17 min, removed after cooking and placed 
inverted onto cooling racks for at least 2 hr post bake.  After cooling, the cakes were 
 109
turned upright and the tops lightly dusted with cake flour to ensure that the rapeseed 
used to determine cake volume did not stick to the cake tops.  The remaining volume 
of the cake pan was filled with rapeseed and leveled off using a small metal spatula 
until the rapeseed was flush with top of the pan.  The pre-determined volume of the 
pan was then recorded, and the rapeseed was slowly measured into a graduated 
cylinder using a funnel.  The volume of the rapeseed was recorded and the cakes were 
saved for TPA analysis.    The cake volume was determined using the formula: 
Pre-determined pan volume ? rapeseed volume = cake volume. 
Whipping Height 
 Whipping height measurements of each of the four treatments were obtained 
by measuring 200 mL of liquid albumen sample into a graduated cylinder and placing 
it into a 5 quart mixing bowl.  The bowl was then attached to a Kitchen Aid Artisan 
Series? mixer with a balloon whisk attachment.  The liquid albumen was whipped 
for 45 s a speed 6, followed by an additional 45 s at speed 10.  Once foam had 
formed, the bowl containing the albumen was removed from the mixture and the 
foam gently leveled off with a spatula to obtain a proper whipping height 
measurement.  A clear plastic ruler was inserted into the foam approximately 0.5 in 
from the center of the bowl and the measurement recorded in millimeters. 
Angel Food Cake TPA 
 The texture profile analysis of the sponge cake samples was done using a 
Stable Micro System?s TA.XT Plus Texture Analyzer (Texture Technologies Corp., 
Marietta, GA).  The TPA function of this equipment measures hardness, sponginess, 
gumminess, resilience, chewiness, cohesiveness, and force.  Once selecting the TPA 
 110
function from the project list, cake samples were placed under a probe attached to a 
cylindrical attachment to ensure an even measurement.  The force at which the cakes 
were compressed is then calibrated using a 5 kg load cell and a 2 kg weight.  Once 
completed, the cakes were compressed twice, taking both the first and second peak 
measurements at 4 cm.  
Statistical Analysis 
The experimental design of this study was a Completely Randomized design 
(CRD).  Statistical analysis was done using the SAS? Statistical Package version 9.0 
(SAS Institute, Cary, NC).  Averages of the samples taken from the sub-replicates 
were analyzed using the General Linear Model (PROC GLM) in SAS and means 
found to be significantly different were analyzed using Fisher?s Protected LSD.   
RESULTS AND DISCUSSION 
   Although the treatments containing triethyl citrate (PW and RW) were 
slightly lower in pH, there was no statistical difference (P<0.05).  Although the 
triethyl citrate products perform well chemically, there are issues concerning 
functionally, which were addressed in this research.  There were no statistical 
differences (P>0.05) in total moisture, fat, or protein between any of the treatments.   
It was discovered that the treatments with triethyl citrate whipping agent 
supplementation were more effective in producing a high volume angel food cake and 
exhibited a positive impact during TPA analysis.  As shown in Figures 1 and 2, the 
two treatments that contained whipping agent were significantly different (P<0.05) 
from the P treatment.  Although there were no significant differences in whipping 
height, it was determined that once baked, the treatments containing the whipping 
 111
agent produced an angel food cake with a greater volume.  It was also determined that 
the PW treatment was a softer cake, having a less gummy and chewy texture.  The P 
treatment produced a gummy, chewy cake that displayed a thicker crust on the top, in 
turn, creating a less desirable angel food cake.  This concurs with the theory that 
pasteurization of albumen, without the addition of an additive to act as a barrier, can 
be detrimental to baking products.  Although the exact reason for this needs further 
research, it can be speculated that triethyl citrate acts as a mechanical barrier which 
protects certain proteins from denaturation during the pasteurization process.   
Conclusions 
The results of this study show that neither pasteurization nor triethyl citrate 
whipping agent supplementation had a true, practical impact on the chemical 
composition of albumen products. The results of the functional portion of this 
experiment revealed that although there were no statistical differences observed in the 
whipping heights of the treatments, the addition of triethyl citrate to albumen 
products created a more voluminous cake post-bake, which exhibited a more 
desirable TPA analysis than those that were left un-supplemented.  More research is 
needed to discover the chemical mechanisms that occur between egg proteins and 
triethyl citrate.  However, it was evident by the results obtained from the P treatment, 
that pasteurization without the supplementation of a whipping agent has a negative 
impact on the functionality of albumen.  
 112
Figure 1. Angel Food cake volume 
 
Angel Food Cake Volume
0
100
200
300
400
PRPWRW
Treatment
Vo
lume (
m
L)
(P<0.05) 
 113
Figure 2. Angel Food cake texture profile analysis 
 
Angel Food Cake TPA
0
200
400
600
PRPWRW
Treatment
grams
Force Springiness Cohesiveness
Gumminess Chewiness
 
(P<0.05)
 114
SUMMARY OF RESULTS 
 
 Although it may seem that the sets of experiments conducted in this 
dissertation are not related, in reality they are not distant relatives. The 
microbiological research, which comprises the first two experiments, studied the 
natural occurrence of the organism, Salmonella Enteritidis.  This is the pathogen most 
commonly associated with eggs and egg products.  Although we tested for other 
bacteria, the recovery of this specific organism was the primary target. 
  The study was conducted over a two year period in which the first 12 months 
acted as the pre-molt period and the second 12 months served as the molt and post 
molt portion of the research.  One of the goals was to observe whether an increase in 
hen age had an effect on the total microflora counts, as well as the incidence of SE.  
For each of the experiments, the exterior shell, interior shell including membranes 
and matrices, and contents containing both yolk and albumen portions were tested.  
Although we utilized two different hen strains for this research, it was discovered that 
only hen age affected the total microflora counts of each of the egg components.  
Also, there was no incidence of SE observed throughout the entire study of the shell 
eggs, and also none found in the environmental samples.  During the molt and post 
molt portion of the study, three molting treatments were used.  No Salmonella 
samples were discovered in the feed-restricted treatment, with the non-molted and 
non-fasted treatments showing the only signs of Salmonella spp., none of which were
 115
 SE.  The second set of experiments dealt with pasteurized liquid products.  Over 30% 
of the eggs marked today are sold in this form, mainly because of their relationship to 
the previous study.  When eggs are pasteurized, they are virtually bacteria free.  This 
creates a safer product for the consumers, but leaves questions as to how they 
function in food products.  The results of this study indicated that although an acidic 
supplement was added to both the whole egg and albumen products, there were no 
significant differences (P>0.05) in the pH between any of the treatments analyzed.  
This was also the case for the chemical analyses performed on both products.  We 
discovered no differences in total fat, total moisture, or total protein.  It was also 
observed that those products that were supplemented with the acidic products (citric 
acid and triethyl citrate) created more voluminous sponge and angel food cakes, 
which exhibited the best overall texture profile analysis.  The whole egg pasteurized 
and whole egg pasteurized with citric acid products also exhibited a stronger gel 
bloom and created custard products with less weep.   
 These results indicate that although eggs are pasteurized to protect consumers 
from possible contact with bacteria, the actual chance of contracting SE from shelled 
eggs is very low, as none was discovered over the course of our two year study.  
However, if consumers are choosing to buy pasteurized products over shelled eggs, it 
would be better to choose those products supplemented with either citric acid or 
triethyl citrate whipping agent, as they provide a more functional use in baking than 
do un-supplemented products
 116
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APPENDICES 
Ashing (Proteins) 
 
Supplies Needed 
 
Phoenix System by CEM 
189 Quartz fiber ashing crucibles 
378 Crucible liners (disks) 
1 set large tongs to transfer crucibles to and from oven 
1 set small tongs or tweezers 
Multiple pairs of Nitrile Laboratory Gloves 
1 set of Phoenix System Gloves provided by CEM 
Scale that measures to 0.0001 grams 
Eggs for sampling 
30 small Turkey Basters 
1 small plastic lunch tray 
 
Procedure 
 
1. Program the Phoenix System for 20-35 minutes and 550?C and allow the 
ashing furnace to pre-heat to 550?C. 
 
2. With Nitrile gloves on and using small tongs, weigh a crucible with 1 disk 
to the nearest 0.0001 mg and record the weight. 
 
3. Tare the scale. 
 
4. Weigh 5.0 grams of sample to the nearest 0.0001 mg into the tared 
crucible and record the weight of the sample. 
 
5. Wearing the Phoenix System gloves, place the crucible with sample and 
disks in the furnace using the large tongs and ash for 20-35 minutes. 
 
6. Remove the crucible from the furnace using the large tongs and place 
them onto the small lunch tray immediately after removal.  The samples 
will be cooled by the time all the samples are removed from the furnace.
 131
 
7. Immediately re-weigh the crucible containing the ash and the disks to the 
nearest 0.0001 mg and record the weight. DO NOT leave the crucibles on
 the counter to cool; they must be weighed immediately to avoid any 
uptake of  moisture from the environment. 
 
  
8. Calculate the % ash in an Excel spreadsheet by using the formula: 
 
 % Ash = Post ashing weight ? crucible and disk weight  X 100 
    Sample weight
 132
Moisture and Fat 
 
Supplies Needed 
 
Multiple pairs of Nitrile Gloves 
1 set of small tweezers 
189 CEM weigh pads 
Eggs for Sampling 
30 small Turkey Basters 
189 CEM plastic sheets 
 
Procedure 
 
1. Press enter to get the Smart Trac out of sleep mode. 
 
2. Press # 3 for ?Load Method? 
 
3. Choose either #1 for Albumen samples or # 2 for Whole Egg or Yolk samples. 
 
4. Once a method is chosen, select ?Ready? to run a test. 
 
5. Choose the ?Sample ID? option and type the name of the sample to be run, 
pressing ?Ready? when finished typing. 
 
6. Wearing a pair of Nitrile gloves, with tweezers, place 2 CEM weigh pads on 
the CEM balance inside the machine and close the lid. 
 
7. Press ?Tare? to tare the pads on the balance. 
 
8. Once tared, place 2.0 and 2.5 grams of sample evenly onto the pads and 
record the weight onto the prepared spreadsheet.  Close the lid and allow the 
test to run (approx 5 min for albumen samples and 10 min for whole egg or 
yolk). 
 
9. Once test is complete, the screen will read the moisture content of the sample.  
Record this % Moisture reading onto the spreadsheet.   
 
10. Remove the sample from the CEM with tweezers and roll out 1 sheet of 
plastic wrap from the CEM station.
 133
 
 
11. Place the sample on the plastic wrapper and roll the sample length wise until 
you have a tube shaped sample. 
 
12. Place the sample and plastic wrap into the holding tubes and compress using 
the compression tool provided by CEM. Be careful not to push the sample out 
of the end of the holding tube. 
 
13. Remove the oil standard from the Smart Trac and replace with the holding 
tube containing the sample. 
 
14. Press ?continue? to run the test. 
 
15. Once test is complete the screen will give you the fat %. Record the % Fat 
onto the spreadsheet and discard the sample by pushing it out of the holding 
tube with the instrument provided with the machine.
 134
 
Albumen Whipping Height 
 
Supplies Needed 
 
6 5 quart Artisan style Kitchen Aid mixers 
12 5 quart Kitchen Aid mixer bowls 
12 Kitchen Aid balloon whisks 
6 clear plastic 12 inch rulers 
6 timers 
12 250 mL graduated cylinders 
6 rubber tipped spatulas 
16.2 liters of liquid, room temperature albumen (10.8 liters of unpasteurized albumen, 
5.4 liters of pasteurized albumen, 5.4 liters of pasteurized albumen with whipping 
agent.) 
 
Procedure 
 
1. Before adding sample, allow the Kitchen Aid mixer with balloon whisk 
attachment to run for 1 min. at speed 10. 
 
2. Measure 200 mL of room temperature albumen sample into a graduated 
cylinder and place in the mixer bowl. 
 
3. Whip the albumen for 45 seconds at speed 6, followed by 45 seconds at 
speed 10 to get a stiff peak foam. 
 
4.  Remove the whisk and mixer bowl from the mixer. 
 
5. Level the foam with the rubber tipped spatula, making sure not to crush or 
disrupt the foam. 
 
6. Insert a 12 inch clear ruler into the center of the bowl and measure the 
foam height to the nearest millimeter. 
 
7. Record the measurement on the prepared Excel spreadsheet.
 135
Angel Food Cakes 
 
Supplies Needed 
 
108 Teflon coated cake pans with volume inscribed on side 
6 Artisan style 5 quart Kitchen Aid Mixers 
12 5 quart Kitchen Aid Mixer bowls 
12 Kitchen Aid balloon whisks 
6-12 hand whisks 
1 sharpie marker 
1 roll colored scientific tape 
1 package post-it notes 
6 rubber tipped spatulas 
6.48 liters of liquid albumen each week 
32.4 grams salt each week 
97.2 grams cream of tartar each week 
6624 grams (approx 14.6 lbs) sugar each week 
2376 grams (approx 5.2 lbs) cake flour each week 
72 mL Vanilla each week 
2 sifters 
4 bowls for sifted materials 
20 weigh boats for measuring 
12 250 mL graduated cylinders 
1 10 mL graduated cylinder 
 
Ingredient List 
 
Homogenized Albumen    180 mL 
NaCl      0.9 grams 
Cream of Tartar     2.7 grams 
Fine Sugar      138 grams 
Vanilla      2 mL 
Granulated Sugar     46 grams 
Cake Flour      66 grams 
 
Procedure 
 
**** Pre-Heat the Oven to 350?F before beginning procedure. 
 
1. Gently hand shake the bags containing the albumen samples for 30 seconds.
 136
 
2. Sift the cream of tartar and NaCl together. 
 
 
3. Place 180 mL of room temperature albumen into the mixer bowl and add the 
sifter NaCl and cream of tartar mixture. 
4. Mix at speed 10 for 45 seconds. 
 
 
5. Add the fine sugar to the batter in 3 increasingly larger portions, mixing at 
speed 6 for 4 seconds after each addition. 
 
 
6. Add 2 mL vanilla while mixing at speed 6 for 4 seconds. 
 
 
7. Sift together the granulated sugar and cake flour twice. 
 
  
8. Add the sifted cake flour and sugar mixture to the batter in 3 equal portions.  
After each addition, fold the mixture into the batter using the hand whisk with 
less than 20 strokes for each addition. 
 
 
9. Weigh 75 grams of batter into a tared cake pan and label the pan using the 
scientific tape to denote which sample it is.  Batter will make 3 cakes. 
 
 
10. Place pans into the pre-heated oven and bake for 17 min. 
 
 
11. Remove cakes from the oven and invert onto a cooling rack for at least 2 
hours. 
 
 
12. After cooling turn the pans upright and dust the tops of the cakes lightly with 
cake flour. 
 
 
13. Record the volume in mL that is engraved on the side of the cake pan on the 
prepared Excel spreadsheet. 
 
 
14. Fill the pan with rapeseed until the top is completely covered and level the pan 
using a ruler or spatula until the rapeseed is flush with the top of the pan. 
 137
15. Measure the rapeseed into a graduated cylinder with a funnel, and record the 
volume in mL on the spreadsheet. 
 
 
16. DO NOT discard cakes after seed volume is taken.
 138
TPA Measurement for Angel Food and Sponge Cakes 
 
Supplies Needed 
 
Texture Analyzer 
Spatula 
Garbage Can 
 
Procedure 
 
 
1. Turn on the Texture Analyzer by flipping the switch on the back of the 
base of the machine. 
 
 
2. Open the Texture Analyzer program icon located on the desktop. 
 
 
3. Login to the program using MCKEE as the username and MCKEE as the 
password. 
 
 
4. Click the ?Project List? icon 
 
 
5. Choose the ?Personal Projects? option from the list. 
 
 
6. Click the ?TPA? option. 
 
 
7. Next, choose the ?TA? option from the menu at the top of the screen. 
 
 
8. Scroll down that list and choose ?calibrate force?. *** Make sure the 
proper load cell is in the machine (5 kg). 
 
 
9. After calibration, choose the ?Run a Test? option.
 139
 
 
10. On the test screen, choose ?Path?? Browse ? My Documents ? and 
create a new folder with the date of the test ? open the folder. 
 
 
11. Make sure auto save is checked to ensure that your data will be saved. 
 
 
12. Place the cake on the machine, and run the test. 
 
 
13. After the test is complete, the cake can be discarded.
 140
Custards 
 
Supplies Needed 
 
81 4 oz ramekins 
81 100 mL glass beakers 
6 Kitchen Aid Mixers 
12 mixer bowls 
12 balloon whisks 
6 timers 
1 roll parafilm 
4 large baking pans 
81 plastic storage containers 
5 boxes of 100 whatman papers 
1 sharpie marker 
Post-it notes 
Baking racks 
Saucepans 
6.75 liters of whole egg 
13.5 cups sugar 
40.5 teaspoons vanilla 
6.75 teaspoons salt 
81 cups milk ( approx. 6 gallons milk) 
1 can Pam Cooking Spray 
6-12 graduated cylinders 
Measuring cups 
 
Procedure 
 
1. Pre-heat oven to 350?C. 
 
2. In a mixer bowl, add 250 mL of whole egg, .5 cups sugar, 1.5 teaspoons 
vanilla, and .25 teaspoon of salt. Blend for 1 min at speed 4 with the 
Kitchen Aid mixer. 
 
3. Heat milk in a saucepan until very hot, stirring occasionally so milk does 
not stick to the pan. 
 
4. With the mixer on speed 4, slowly add 3 cups of heated milk, turning off 
the mixer once all milk has been added.
 141
 
5. Lightly spray 3 ramekins with Pam spray and label using a sharpie marker. 
 
6. Add 100 mL of the mixture into the ramekin. 
 
7. Using 3 100 mL beakers, place 60 grams of the mixture into the beakers. 
 
8. Place beakers and ramekins into a large baking pan and fill with very hot 
water to within ? inch of the top of the ramekins. 
 
9. Bake at 350?C for 40 min. 
 
10. While custards are baking, place 5 whatman papers on a tared container 
lid and record the weight to the nearest 0.0001 gram.  Place a post-it note 
with the weight of the papers on the top of the lid of the plastic container. 
 
11. Remove the beakers and ramekins from the oven and place on a wire 
cooling rack until they reach room temperature. 
 
12. Once the beakers reach room temperature, cover the tops of the beakers 
with parafilm and place in the walk-in cooler (4?C) for 18 hours. 
 
13. When the ramekins reach room temperature, gently run a knife around the 
edge of the ramekin to loosen the custard.  Invert the ramekin over the 
whatman papers until it drops in the center of the paper.  Place the lid over 
the container with the custard and place in the cooler for 18 hours. 
 
14. After 18 hours, remove the custards in the plastic containers from the 
cooler and transport to the lab.  Gently remove the lid and discard the 
custard in the garbage can, making sure not to tear the paper.  Weigh the 
paper to the nearest 0.001 gram to determine the amount of weep from the 
custard. 
 
15. Remove the beakers from the cooler and perform Bloom test using texture 
analyzer.
 142
Bloom for Custards 
 
Supplies Needed 
 
Texture Analyzer 
Spatula 
Garbage Can 
 
Procedure 
 
 
1. Turn on the Texture Analyzer by flipping the switch on the back of the base of 
the machine. 
 
 
2. Open the Texture Analyzer program icon located on the desktop. 
 
 
3. Login to the program using MCKEE as the username and MCKEE as the 
password. 
 
 
4. Click the ?Project List? icon 
 
 
5. Choose the ?Personal Projects? option from the list. 
 
 
6. Click the ?Bloom? option. 
 
 
7. Next, choose the ?TA? option from the menu at the top of the screen. 
 
 
8. Scroll down that list and choose ?calibrate force?. *** Make sure the proper 
load cell is in the machine (5 kg). Follow directions on screen. 
 
 
9. After calibration, choose the ?Run a Test? option.
 143
 
 
10. On the test screen, choose ?Path?? Browse ? My Documents ? and create 
a new folder with the date of the test ? open the folder. 
 
 
11. Make sure auto save is checked to ensure that your data will be saved. 
 
 
12. Place the custard on the machine, and run the test. 
 
 
13. After the test is complete, the custard can be discarded.
 144
 
Sponge Cakes 
 
Supplies Needed 
 
6 Kitchen Aid Mixers 
12 mixer bowls 
12 balloon whisks 
6 250 mL glass beakers for weighing egg 
6 hand whisks 
12 graduated cylinders 
1 10 mL graduated cylinder 
81 Teflon coated cake pans 
1 roll scientific tape 
Sharpie markers 
Post-it-notes 
3726 grams whole egg (approx 8.2 lbs) 
2413.8 grams sugar (approx 5.3 lbs sugar) 
54 mL vanilla 
1387.8 grams cake flour (approx. 3.05 lbs) 
 
Procedure 
 
1. Pre-heat oven to 350?C. 
 
2. Stomach whole egg for 30 seconds at 200 rpm to homogenize. 
 
3. Place 138 grams whole egg and 89.4 grams sugar into the mixer bowl. 
 
4. Mix for 15 seconds at speed 2, then for 4 min. at speed 10. 
 
5. Add 2 mL vanilla and mix at speed 6 for 4 seconds. 
 
6. Add the cake flour to the batter in 4 equal portions, folding in after each 
addition with less than 20 strokes. 
 
7. Weigh 75 grams of batter into a tared cake pan. 
 
8. Place pans in the oven and bake for 17 min.
 145
 
9. Remove the pans from the oven and invert on a cooling rack for at least 2 
hours. 
 
10. After cooling, turn the pans upright and lightly dust the tops with flour. 
 
11. Fill the pan with rapeseed until the top is mounded and then level off until 
the seed is flush with the top of the cake pan. 
 
12. Record the volume on the side of the pan. 
 
13. Measure the rapeseed into a graduated cylinder by using a funnel and 
record the volume. 
 
14. DO NOT remove the cakes from the pans after rapeseeding. 
 
15. Save cakes for TPA analysis on the texture analyzer.