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Egg Proteins
Snigdha Guha
a
, Kaustav Majumder
a
, and Yoshinori Mine
b
,
a
Department of Food Science and Technology, University of Nebraska-
Lincoln, NE, United States; and
b
Department of Food Science, University of Guelph, Guelph, ON, Canada
© 2018 Elsevier Inc. All rights reserved.
Introduction 1
Egg Shell Proteins 2
Egg White Proteins 3
Ovalbumin 3
Ovotransferrin 4
Ovomucoid 5
Ovomucin 5
Lysozyme 5
Minor Proteins in Egg White 6
Ovoglobulin 6
Cystatin 6
Avidin 6
Ovoflavin 7
Egg Yolk Proteins 7
Low-Density Lipoprotein (LDL) 8
High-Density Lipoprotein (HDL) 8
Phosvitin 8
Livetin 8
Conclusion 9
References 9
Further Reading 12
Glossary
Amphipathic a chemical compound containing both hydrophobic/lipophilic and hydrophilic properties
Apolipoproteins proteins that specifically bind lipids to produce lipoproteins. Their major role is to carry lipids through the
circulatory and lymphatic system
Calcification impregnation with calcium or calcium salts
Glycation formation of a covalent bond between a sugar molecule (such as fructose or glucose) to a protein or lipid molecule
Isoelectric point refers to the pH at which a specific molecule bears no net electrical charge
Lectin any protein or glycoprotein that is capable of binding to the sugar moieties of glycoproteins and glycolipids present on
the surface of cells in most organisms. These proteins help in stimulating lymphocyte proliferation
Polymorphism refers to the branching of the genetic tree which is because of two or more alleles present at one DNA position
or in one DNA region, each with significant frequency in the population
Reactive oxygen species (ROS) chemical species which contains oxygen and are chemically reactive, for instance, superoxide,
peroxides, singlet oxygen
Nomenclature
TSAA Total Sulfur Amino acids, i.e. (Methionine þCystine)
Quercetin Plant polyphenol belonging to the flavonoid group and has a bitter taste. It is present in many vegetables, fruits,
grains and leaves
Introduction
The chicken eggs have always been recognized as an excellent source of human nutrition, especially for the dietary protein. The
chemical composition of an egg has always fascinated human mind, may be due to the compartmentalized structure of the whole
1
egg. However, only in recent years, with the help of innovative research tools, scientists have revealed the structural and functional
diversity of the different components in eggs. These revelations diversify the use of eggs in various processed food products and
increase the commercial value of an egg. Egg proteins are well-known for its functional properties and are massively used as an
ingredient to enhance the texture or flavor of variety of food products. Additionally, egg proteins are also a potential source of bioac-
tive proteins and peptides. The bioactive egg proteins and peptides can exhibit health beneficial effect above and beyond their
known nutritional value. Research of last two decade has highlighted some bioactivity of egg proteins and broadened the use of
egg as a critical ingredient of functional foods or nutraceuticals. Subsequently, egg proteins, especially proteins from egg white,
are known as a major allergen affecting nearly 2.5% young children in the USA (Caubet and Wang, 2011). Therefore, this chapter
briefly discusses the different proteins of egg and their functional and biological properties.
The egg consists of three major portions; a) eggshell, b) egg white, and c) egg yolk. Protein is one of the major component present
in all three parts of the egg, and egg white is the prime source of proteins. The following section discusses the major proteins found
in each part of the egg.
Egg Shell Proteins
The eggshell is the outermost layer of an egg and is mainly composed of a foamy layer of cuticle, a calcium carbonate layer, and then
two flexible membranes (inner and outer membrane). This whole structure together retains the egg white or albumen and egg yolk
inside the egg and also prevent the invasion of any pathogenic bacteria (Burley and Vadehra, 1989).
The organic matter of chicken eggshell and eggshell membrane comprises of a complex mix of proteins and polysaccharides, out
of which proteins constitute almost 70% of the total organic matter (Tullet, 1987). The eggshell membrane has been found to
contain many bacteriolytic enzymes, such as N-acetylglucosaminidase and lysozyme, and other components which might have
a role in preventing the invasion of Gram-negative and Gram-positive bacteria. Moreover, the eggshell membrane hydrolysates
have been found to contain hydroxyproline suggestive of the presence of collagen in the membrane layers (Nakano et al.,
2003), the collagen constitutes almost 10% of the total proteinaceous matter in the eggshell.
Eggshell mainly comprises of various ubiquitous proteins which are extensively expressed in different organs. Osteopontin,
a phosphorylated glycoprotein, is found in kidney, bones and many body secretions and it was found to be expressed in the uterus
epithelial cells during the calcification of the eggshell (Pines et al., 1994). This protein is mainly confined to the mammillae, core of
the non-mineralized shell membrane fibers, and the outermost segment of the shell palisade layer (Fernandez et al., 2003). Osteo-
pontin inhibits the precipitation of calcium carbonate in the eggshell, and the protein loses its inhibitory activity upon dephosphor-
ylation with alkaline phosphatase. This suggests that osteopontin might have a role as a modulator in the precipitation of calcium
carbonate in the uterine fluid or even as an inhibitor during the termination of the calcification process (Hincke and St Maurice,
2000).
Clusterin is another ubiquitous eggshell secretory protein which is a heterodimeric glycoprotein bonded with disulfide linkages
(Mann et al., 2003). This particular protein is found in many tissues and is also present in all the calcified regions of the eggshell. In
the uterus, it is secreted by the tubular gland cells into the uterine fluid, irrespective of the shell calcification stage. Clusterin might
have a role in preventing premature aggregation and precipitation of eggshell proteins by acting as an extracellular chaperone in the
uterine fluid (Mann et al., 2003). There are specific proteins which are unique to the eggshell and are secreted only by the tissues
present in the shell. These proteins have been identified in domestic hens only.
Ovocleidin-17 (OC-17) is a 142 amino acid long phosphorylated protein having a C-type lectin domain, and it was the first
protein to be purified to homogeneity. This protein is also present as a 23 kDa glycosylated protein in a minor form (Mann,
1999). The tubular gland cells of the uterus secrete OC-17 throughout the calcified part of the shell during the entire calcification
period (Hincke et al., 1995;Mann and Siedler, 1999;Reyes-Grajeda et al., 2004). OC-17 modifies the shape of calcium carbonate
crystals in vitro (Reyes-Grajeda et al., 2004).
Ovocleidin-116 (OC-116) is an 80 kDa protein with 742 amino acids, and it was the first eggshell protein which was cloned
(Hincke et al., 1999). The protein comprises of two disulfide bonds and two N-glycosylations (Mann et al., 2002). OC-116 was
named ovoglycan (Fernandez et al., 2001,2003) and it forms the protein core of the primary proteoglycan of the shell (Carrino
et al., 1997). The carbohydrates of OC-116 comprises of 17 different oligo-structures (Nimtz et al., 2004). Eight of them were
hybrid-type, four were the high-mannose type, and the rest five had a complex-type structure. When present in the uterine fluid,
OC-116 exists as a 116 kDa protein due to glycosylation modification and as a 190 kDa protein after glycation (Arias et al.,
1992;Fernandez et al., 1997). This protein is thought to have a role in the modulation of calcite growth.
Ovocalyxin-32 (OCX-32) is released in the uterine fluid by the surface epithelial cells of the uterus during the end of the calci-
fication phase and thus it is mainly confined to the outer regions of the shell, i.e., the vertical crystal layer, cuticle, and the palisade
layer (Gautron et al., 2001b,2003;Hincke et al., 2003). Therefore, it has been suggested that this protein has a role in the termi-
nation of the calcification process of the eggshell.
Ovocalyxin-36 (OCX-36) is found in abundance in the uterine fluid during the calcification process, and the expression of this
protein is highly upregulated. This protein has been cloned as well (Gautron et al., 2007). It has been reported that OCX-36 has
homologous similarities with proteins involved in innate immune responses such as bactericidal permeability increasing proteins,
lipopolysaccharide binding proteins, and Plunc family proteins, which suggests the fact that OCX-36 may also be involved in the
defence mechanisms to keep the egg pathogen free (Gautron et al., 2007).
2Egg Proteins
Ovocalyxin-25 and -21 are also two other eggshell proteins which are exclusively detected in tissues undergoing mineralization.
Database analysis reported that Ovocalyxin-21 has remarkable homologies with brichos domain-containing proteins. The Brichos
domain consists of nearly 100 amino acids and comparing the similarities, several functions of the ovocalyxin-21 proteins were
postulated including chaperon-like functions (Sanchez-Pulido et al., 2002). Ovocalyxin-25 contains two protease inhibitory
domains, one of which is the WAP-type. The matrix protein from the nacreous layer of the pearl and shell of molluscs, known
as lustrin A, also has the same inhibitory WAP-type domains (Shen et al., 1997).
Egg White Proteins
The egg white is made up of four individual layers: chalaziferous layer, thin layer, thick layer, and the chalazae cord. The thin layer
accounts for about 23.3% of egg white, which is further separated into two layers, i.e., inner and outer thin layers. The thin inner
layer (16.8% of egg white) is attached to the chalaziferous layer, which accounts for about 2.7% of egg white, whereas, the thin outer
layer is connected to the inner eggshell membrane. The outer and inner thin layers are separated by the thick or viscous layer which
accounts for the most substantial portion of egg white, i.e., 57.3% (Brake et al., 1997;Conrad and Philips, 1938;Li, 2006).
Water is the primary constituent of egg white which accounts for about 84% to 89% of the total egg white or albumen weight.
Among albumen solids, proteins are the major constituents (10%–11%), while the minor components include carbohydrates
(0.9%), lipids (0.03%), vitamins and minerals (Li-Chan and Nakai, 1989).
Egg proteins are well known for their high nutritional quality, excellent digestibility and comprise of all the essential amino acids
necessary for the human nutrition and development (Friedman, 1996). Egg albumen consists of several different protein compo-
nents which have been identified and characterized through modern high-resolution analytical techniques (Raikos et al., 2006). In
a study, 78 egg white proteins were detected using 1-dimensional electrophoresis and liquid chromatography tandem mass-
spectrometry (LC-MS/MS) (Mann, 2007). However, among egg white proteins, ovalbumin, ovotransferrin, ovomucoid, ovomucin,
and lysozyme have been studied extensively due to their abundant presence in egg albumen. Physicochemical characteristics of
major egg white protein is provided in Table 1. The structure and chemical composition of these proteins are described in details
in the following section.
Ovalbumin
Ovalbumin constitutes about 54% of the total egg albumen and thus it is the primary protein present in egg white. It is a phosphor-
ylated glycoprotein made up of complete three subunits having different phosphate groups along with a carbohydrate group
attached to its N-terminal (Li-Chan et al., 1995). Ovalbumin is a member of the serpin (serine protease inhibitors) superfamily
despite lacking the inhibitory activity unlike the other serpin-like proteins (Huntington and Stein, 2001). The molecular weight
of ovalbumin is 45 kDa and is composed of 386 amino acids along with two genetic polymorphisms seen at 290 (Glu/Gln)
and 312 (Asn/Asp) (McReynolds et al., 1978). Among all other egg albumen proteins, ovalbumin is a unique protein as it
Table 1 Physio-chemical properties of major and minor egg white proteins
Protein dry weight of albumen (g/kg) Isoelectric point (PI) Molecular weight (kDa) T
d
(C)
Major proteins
Ovalbumin 540 4.5 45 84
Ovotransferrin 120 6.1 76 61
Ovomucoid 110 4.1 28 77
Ovomucin 35 4.5–5.0 5500–8300 –
Lysozyme 34 10.7 14.3 75
Minor proteins
Ovoglobulin (6.1–5.3)
G
2
globulin 40 5.5 30–45 92.5
G
3
globulin 40 4.8 ––
Ovoinhibitor 15 5.1 49 –
Ovoglycoprotein 10 3.9 24.4 –
Ovoflavoprotein 8 4.0 32 –
Ovomacroglobulin 5 4.5 769 –
Cystatin 0.5 5.1 12.7 –
Avidin 0.5 10 68.3 –
‘–‘ represents not determined.
Modified from Powrie, W.D., Nakai, S., 1985. Characteristics of edible and fluids of animal origin: egg. In: Fennema, O. (Ed.), Food
Chemistry. New York, Marcel Dekker, pp. 829–855 and Data compiled from Li-Chan, E.C.Y., Powrie, W.D., Nakai, S., 1995.
Egg Proteins 3
contains six cysteine residues, two of which are involved in a disulfide bond between Cys74 and Cys121, while the rest four include
free sulfhydryl (eSH) groups. One out of the four eSH group is reactive only when the protein is denatured, however, the other
three are masked in the native state (Fothergill and Fothergill, 1970).
The amino acid composition analysis of egg albumen revealed that 50% of the total amino acids are hydrophobic while 30% are
acidic and charged amino acids, the latter contributing to the acidic isoelectric point (pI) of 4.5. Ovalbumin has a unique amino
acid distribution as compared to other glycoproteins. It lacks an N-terminal ladder sequence but contains an acetylated glycine and
proline in the N- and C-terminal, respectively, along with carbohydrate moiety attached to the amino acids in the N-terminal
(McReynolds et al., 1978;Huntington and Stein, 2001).
Ovalbumin-Y is a chimeric glycoprotein with an amino acid sequence very similar to the native ovalbumin and a carbohydrate
group identical to ovomucoid (Hirose et al., 2006). Ovalbumin-Y protein was first identified and characterized by Nau et al. (2005)
by 2-Dimensional PAGE and peptide mass fingerprinting. Ovalbumin Y is not phosphorylated unlike ovalbumin; however, it is
glycosylated. Three isoforms of ovalbumin Y protein and five isoforms of ovalbumin-related Y protein have been identified via elec-
trophoresis, each protein differing in their pIs. However, this polymorphism could not be explained by genetic variations or by
phosphorylation or glycosylation levels (Guèrin-Dubiard et al., 2006).
Ovalbumin in its native form is resistant to digestion by trypsin but once it is heat denatured or given an acid or pH change
treatment, it becomes susceptible to trypsin digestion. With high pH and temperature dependent denaturation, ovalbumin converts
into a thermally stable form known as S-ovalbumin (Pelegrine and Gasparetto, 2006). Sugimoto et al. (1999) demonstrated that
storage temperature influences this conversion more than the storage time, where longer storage time at 20–25 C did not affect the
conversion. However, Huang et al. (2012), demonstrated that during storage, the conversion of ovalbumin to S-ovalbumin had
been attributed to an increase of pH and change of pH has a direct effect on the conversion rather than temperature. Therefore,
despite temperatures of 4 C, long time storage of eggs may increase the conversion of native ovalbumin to S-albumin by 81%
(Huang et al., 2012).
Ovalbumin is also the primary allergen in egg white which is responsible for IgE-mediated allergic reactions (Caubet and Wang,
2011). The ovalbumin epitopes which bind IgE consists of mainly polar, charged and hydrophobic amino acids and these
sequences are mostly made up of b-sheet and b-turn structures. The only allergenic epitope which comprises of an alpha helix is
Asp95-Ala102 (Kim, 2002).
Ovotransferrin
Ovotransferrin is a monomeric glycoprotein which is involved in the transfer of ferric ions to the developing embryo from the
hen’s oviduct. Ovotransferrin accounts approximately 12% of the entire egg white protein (Desert et al., 2001;Abdallah and El
Hage Chahine, 1998). Ovotransferrin belongs to the transferrin protein family and has been reported to have around 50%
homology with mammalian lactoferrin and transferrin (Mazurier et al., 1983). Ovotransferrin can bind 2 mol of different
metal ions per mole of protein. For, ovotransferrin, lactoferrin and serum transferrin, each lobe binds one carbonate anion
and one Fe
þ3
atom (Lambert et al., 2005). Such iron complex formation in ovotransferrin inhibits microbial growth that
requires iron.
The N and C lobes of ovotransferrin consist of a single iron-binding site located in a deep cleft along with 15 disulfide bridges
which maintain the globular structure of the protein (Kurokawa et al., 1999). The N- and C- terminal lobes of ovotransferrin form
two sulfide bonds where Ala1-Tyr72 in the N-terminal segment acquires a local-native like confirmation (Mizutani et al., 1997).
This interaction between the two lobes is very critical for iron acquisition (Alcantara and Schryvers, 1996). The various metal-
and anion-binding properties of the iron binding sites of ovotransferrin can be attributed to the presence or absence of basic amino
acid residues (Nadeau et al., 1996).
The process of in-vitro Fe
þ3
uptake and release by ovotransferrin are reported to be very similar, but not identical, to lactoferrin
and serum transferrin. The Fe
þ3
is bound very tightly by the four protein ligands (Tyr92, Asp60, His250 and Tyr191 present in the N-
lobe) in a closed interdomain cleft (Abdallah and Chahine, 1999). Ovotransferrin efficiently binds Fe
þ3
at pH greater than 7 and
releases any bound Fe
þ3
at a pH lesser than 4.5 (Guèrin-Dubiard et al., 2006). On uptake of Fe
þ3
, the transferrins undergo a major
conformational transition from the apo structure (open-form/iron-free) to the closed/iron-bound holo formation, suggesting that
initial binding occurs in the open form (Mizutani et al., 1999). Both these forms have significantly different physiochemical prop-
erties, for instance, the holo-form emits a salmon pink color due to the presence of iron whereas the apo-form does not have any
color. Moreover, the apo-form is more prone to physical and chemical changes as compared to the holo-form (Kurokawa et al.,
1999).
Apart from the iron-binding capacity, several recent studies have investigated other structural and functional features of ovo-
transferrin that might be associated with various biological properties. For instance, the embryos of mammal and bird’s egg are
susceptible to oxidative stress, and thus maintenance of a constant, reducing environment during the development of the embryo
can provide protection and although much knowledge is not present currently, however egg white is suggested as the primary target
for this. Ibrahim et al. (2006) reported that ovotransferrin is capable of autocleavage at specific sites once it gets reduced by thiol
reducing agents. This autocleavage occurs due to a unique chemical reaction between the four tripeptide motifs present on both
sides of the two disulfide domains (115–211 and 454–544 residues) of ovotransferrin protein. It has been found that many
auto-processing proteins contain these reduction-scissile sequences (His/Cys-X-) which suggest that this sequence is evolutionarily
conserved.
4Egg Proteins
Ovomucoid
Ovomucoid is a glycoprotein which belongs to the Kazal family of protein inhibitors. It constitutes 11% of the total egg white
proteins and is thermally stable (Li-Chan and Nakai, 1989). The protein consists of 186 amino acids with a molecular mass of
28 kDa (Kovacs-Nolan et al., 2000). It consists of 9 disulfide bonds and has three different domains which are crosslinked only
by the intra-domain disulfide bonds. Ovomucin has trypsin inhibitory effect, and the active site for the trypsin inhibitory activity
lies within the Domain II of the protein. However, considerable variations in the inhibitory activities and specificities of the
domains have been reported from different avian species (Li-Chan and Nakai, 1989). Chicken ovomucoid is one of the significant
egg white allergens, and it plays a crucial role in the pathogenesis of IgE-mediated allergic reactions (Mine and Zhang, 2001,2002a;
Mine and Rupa, 2003a,2004). This allergenic potential could be attributed to its higher stability towards gastrointestinal digestion
and heat treatment (Hirose et al., 2005). Yoshino et al. (2004) reported that the digestibility of ovomucoid by pepsin is much better
over the pH range of 1.5–2.5. However, the digestibility loses at a pH of 3.0 or higher. Moreover, pepsin-digested fractions of ovo-
mucoid retain its trypsin-inhibitory activities. Besler et al. (1997) reported that the epitopes on ovomucoid which were responsible
for the IgE binding were present only on the protein backbone and not the carbohydrate groups. Nine IgE epitopes (5–16 amino
acids) and eight IgG epitopes (5–11 amino acids) were identified within the primary ovomucoid sequence. Through mutational
studies of the epitopes, it was found that charged amino acids (lysine, glutamic acid, and aspartic acid), polar amino acids (cysteine,
tyrosine, threonine, and serine), and hydrophobic (glycine, leucine, and phenylalanine) are crucial for antibody binding (Mine and
Zhang, 2002b). Numerous studies have been conducted to alter the composition and structure of the ovomucoid epitopes respon-
sible for the allergenicity (Mine and Rupa, 2003b). Some of the attempts made include heating in the presence of wheat flour
(Kovacs-Nolan et al., 2000;Kato et al., 2001), gamma irradiation along with heating (Lee et al., 2002), deglycosylation by
endo-beta-N-acetylglucosaminidases (Yamamoto et al., 1998), and genetic modifications (Rupa and Mine, 2006a). However,
none of these modifications made any significant changes to the allergenic epitopes of ovomucoid, suggesting that the epitopes
were extremely resistant to any modifications.
Ovomucin
Ovomucin contributes to about 3.5% of the total egg white proteins. It is a sulfated glycoprotein which is responsible for the jelly-
like structure of egg white. The protein consists of two parts: soluble part (8,300 Da), which is the main component of the inner and
outer egg white, and the insoluble part (220–270 kDa), which is responsible for the insoluble gel-like fraction of thick albumen
(Omana and Wu, 2009). Both fractions are made up of two subunits, a-ovomucin and b-ovomucin, but have different carbohydrate
contents. The soluble fraction consists of 40 a- and three b-subunits, while the insoluble fraction contains 84 a- and 20 b-subunits
(Robinson and Monsey, 1971;Omana and Wu, 2009). There are two distinct subunits of the a-subunit, i.e., a1 and a2, and both the
a-subunits have lesser carbohydrate groups than b-subunits. Acidic amino acids such as glutamic acid and aspartic acids mainly
make up the a-subunit (Omana and Wu, 2009), whereas, serine and threonine primarily make up the b-subunit (Robinson and
Monsey, 1971). A study by Toussant and Latshaw (1999) reported that the quality of eggs could be positively correlated with
the amount of ovomucin present in the thick albumen. However, a higher concentration of highly glycosylated b-ovomucin con-
taining hexoses, sialic acid, and hexosamines, signifies the inferior quality of eggs. Ovomucin is responsible for many of the func-
tional and biological properties of egg white. It has a significant role in thinning of egg white during prolonged storage. Studies have
shown that thinning of egg white can be either due to disruption of the ovomucin-lysozyme complex or the reduction of disulfide
bonds leading to the degradation of ovomucin (Abeyrathne et al., 2014). Moreover, it is known for its exceptional emulsifying and
foaming properties (Mann, 2007).
Lysozyme
Egg white lysozyme consists of 129 amino acids and is a 14.4 kDa protein with a pI of 10.7. Lysozyme present in the egg is unique as
it is highly soluble and stable as compared to lysozyme present in other foods. Although the lysozyme present in egg exists as
a monomer, it is also frequently found as a dimer which leads to its thermal stability. Four unique disulfide bonds stabilize the
tertiary structure of egg lysozyme (Kato et al., 2006). Lysozyme tends to bind to negatively charged proteins in the egg albumen
such as ovalbumin, ovomucin, and ovotransferrin (Abeyrathne et al., 2014). The chalaza and the chalaziferous layer mainly consists
of the lysozyme–ovomucin complex. The complete structure with a resolution of 2 Aand the amino acid composition of lysozyme
had been established in the 1960s (Li-Chan and Nakai, 1989). However, still many research is carried out to investigate its structure
and function further. Three-dimensional structural analysis, at the resolution of 1.46 A, revealed the hexagonal crystal form of lyso-
zyme (Blake et al., 1965;Kato et al., 1992). Various studies have also been conducted to investigate the structural changes of lyso-
zyme induced by different conditions, such as aqueous-organic solvent mixtures (Griebenow & Klibanov, 1996), pH variations
(Babu and Bhakuni, 1997), co-crystallization in presence of different alcohols (Deshpande et al., 2005), sorbitol (Petersen et al.,
2004), in presence of thiol reagents (Raman et al., 1996), and supercritical CO
2
treatment followed by heat treatment (Liu
et al., 2004). The first purification of lysozyme was done using the high concentration of ammonium sulfate, however, it led to
the modification of the characteristics and morphology of the protein due to the high salt concentration used during extraction
(Liu et al., 2004;Abeyrathne et al., 2013). The purification technique which is commonly used nowadays for lysozyme is cation
exchange chromatography which makes use of the high pI value of the protein (Abeyrathne et al., 2013). However, due to the small
Egg Proteins 5
size of the resin granules used in the chromatography, the flow rate is low which makes it a time-consuming process. Crystallo-
graphic structures of some of the three major egg white proteins are listed in Fig. 1.
Minor Proteins in Egg White
Egg white contains more than 50 proteins (Mann, 2007), among which the five major ones are described above. However, there are
some other proteins, which although are present in very small amount, play a crucial role in determining the physicochemical and
structural properties of the egg. Four of such proteins are described in the following section.
Ovoglobulin
Ovoglobulin constitutes 4% of the total egg white proteins and is a minor protein. The protein is made up of two subunits G2 and
G3, with molecular weights of 36 kDa and 45 kDa respectively (Ogawa and Tanabe, 1990;Damodaran and Razumovsky, 1998).
Ovoglobulin was reported to be completely soluble in high and low ionic strength salt solution and coagulated with heat treatment;
these properties were very similar to ovalbumin (Damodaran and Razumovsky, 1998). Ovoglobulin G2, present in chicken eggs,
shows polymorphism (Asal et al., 1993). Ovoglobulin has been reported to be crucial for the foaming properties of egg white, even
though other biological functions are not well known yet (Sugino et al., 1997b). In a study by Damodaran and Razumovsky (1998),
the competitive adsorption of the five major egg white proteins described above, along with ovoglobulin was studied, at low and
high ionic strengths, and it was found that at 0.1 ionic strength, only ovoglobulin and ovalbumin were able to adsorb at the inter-
face, while the other proteins were excluded from the interface.
Cystatin
Cystatin is a cysteine proteinase inhibitor which inhibits the action of thiol proteases, for instance, papain and ficin. Cystatin of the
chicken egg contains two disulfide bonds near the carboxy-terminal along with a reactive site which is highly conserved. The pI of
phosphorylated cystatin is 5.6, whereas it is 6.5 for the non-phosphorylated form (Guèrin-Dubiard et al., 2006), with a molecular
weight of 13kDA (Li-Chan and Nakai, 1989). In a study, the effects of storage conditions of eggs, the age of hens, and heat treatment
of albumen on the cystatin activity was investigated. The cystatin present in eggs laid by hens, either older than 60 weeks or younger
than 30 weeks, had the lowest activity, while eggs laid by hens aged 40–50 weeks had the highest cystatin activity. Furthermore, the
eggs stored for 28 days at 15 C showed a 4%–12% decrease in the cystatin activity. Thermal treatments also decreased the activity
(Trziszka et al., 2004). Apart from having proteinase inhibitor activity, cystatin has been reported to have bioactive properties, such
as, antimicrobial activity against bacterial pathogens (Wesierska et al., 2005), and inhibitor of bone matrix degradation in the
resorption lacunae adjacent to osteoclasts (Brand et al., 2004).
Avidin
Avidin is well known for its biotin-binding activities and is essentially a tetrameric glycoprotein from egg albumen. All the four
monomers of the protein are capable of binding biotin and establishing a strong interaction with a dissociation constant of
10–15 M (Stadelman et al., 1995;Mine and Yang, 2010). The protein constitutes approximately 0.05% of the total proteins in
egg white. Each avidin chain comprises of 128 amino acids which are arranged as eight-stranded antiparallel beta-barrel, where
the D-biotin binding site is defined by the inner regions. The secondary structure of avidin mainly comprises of beta-sheets and
extended beta-turns (66%), while the rest is made up of b-turns and disordered structures (Stadelman et al., 1995). Avidin has
been shown to have insecticidal and antimicrobial activity. The insecticidal activity has been used with genetically engineered
and host plant resistance against Colorado potato beetle (Rupa and Mine, 2006a). Bacteriostatic effect of avidin has been reported
against Salmonella typhimurium. However, the effect is lost on biotin addition (Stadelman et al., 1995). Terminal mannose and
Figure 1 Crystallographic structure of major egg white proteins. Protein Data Bank.
6Egg Proteins
N-acetylglucosamine residues of avidin are capable of binding to lectins. Tumor cells express lectins on their cell surface at various
levels, which acts as an important biomarker. Thus binding of avidin and cytotoxic agents can be easily detected via cell surface
lectins. This mechanism was considered for tumor treatments, and avidin served as a potential vehicle for transport of toxins, drugs,
therapeutic genes, and radioisotopes (Yao et al., 1998).
Ovoflavin
Ovoflavin, also known as riboflavin-binding protein or ovo-flavoprotein, is a phosphoglycoprotein which is present in both egg
yolk and white in equal amounts. This protein is bind with riboflavin or vitamin B2 and the protein contains the highest selenium
(Se) content (1800 ng/g) as compared to other egg proteins (Kiliç et al., 2002). Ovoflavin in egg white constitutes 219 amino acids
with the presence of pyroglutamic acid at the amino terminus (96 Although the biological function of ovoflavin is not fully under-
stood its ability to bind minerals and vitamins suggests that it may be involved in nourishing the embryo with vitamins and
minerals during development (Hamazume et al., 1984).
Egg Yolk Proteins
Egg yolk is composed of plasma and granules, located between the thin and thick albumen, supported by the chalazae. Yolk plasma
constitutes 80% of the yolk fraction, and its protein content is of 23% on a dry basis (Freschi et al., 2011), composed of Low-Density
Lipoprotein (LDL) (15%) and globular glycoproteins (15%) (Laca et al., 2015). Proteins such as livetins present as g-livetins are
mainly IgY antibodies (Chalamaiah et al., 2017), whose functions have been applied as immunological supplements in foods
(Yang et al., 2014) and several other industries. Yolk granules nonetheless have a higher concentration of lipids (33%) and proteins
(58%) in comparison with plasma (Laca et al., 2014). Their structure formation is mainly composed of non-soluble HDL-phosvitin
complexes. Therefore a medium ionic strength has shown to modify its solubility. Protein composition is also dependent on factors
such as feed intake and environment during the hen’s productive life. Molecules such as pigments or vitamins determine the color of
the egg yolk. Studies supplementing herbs, quercetin, and TSAA with lysine, improved the color, oxidative stability, and reduced
yolk protein respectively (Simitzis et al., 2018;Hammershøj and Johansen, 2016;Novak et al., 2004). Also, egg lipid and protein
content vary throughout the productive cycle of the hen. In order to maintain egg weight homogeneity, a reduction in the amino
acid content is realized. Furthermore, alterations in the environment such as heat and feed intake undermine lipid content in the
organism. Heat stress initiates lipid peroxidation in cell membranes due to the release of hormones corticosterone and catechol-
amines (Asli et al., 2007). The following section describes the three major egg yolk proteins and Fig. 2 depicts all the egg yolk
proteins present under reducing and non-reducing conditions.
Figure 2 SDS PAGE profile of egg yolk proteins under non-reducing (NR) and reducing (R) conditions (a and a0) whole EY (b and b0) EY plasma (c
and c0) EY granule (std) MW standard. From Guilmineau, F., Krause, I., Kulozik, U., 2005. Efficient analysis of egg yolk proteins and their thermal
sensitivity using sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing and nonreducing conditions. J. Agric. Food Chem. 53,
93299336.
Egg Proteins 7
Low-Density Lipoprotein (LDL)
LDL proteins are predominant in yolk plasma with an 85% protein composition. Its structure is characterized by a micelle nano-
structure containing triglycerides and cholesterol (Anton et al., 2003;Martin et al., 1964). Emulsification properties of LDL is an
important application for the food industry. Factors such as heat treatment, ionic strength, particle dispersion and defatting induced
protein changes in the structure affecting its functionality (Kiosseoglou, 2003). For instance, amphipathic side chain structure makes
the protein interact with hydrophobic and hydrophilic interfaces such as W/O or O/W. Apolipoproteins in LDL micelle adsorb faster
than low molecular weight proteins, helped by its structure flexibility (Anton et al., 2003;Mine, 1997,1998;Martinet et al., 2003).
In contrast, granules protein has shown an emulsification property dependent of pH. Specifically, at pH 4, its emulsification capacity
is reduced as a result of protein dimer formation (Aluko and Mine, 1998). Due to pH changes and an ionic strength increase, lipo-
proteins tend to minimize the interaction of granules proteins in an emulsion. The delivery mechanism is among other potential
applications of LDL when combined with polysaccharides (Zhou et al., 2016).
High-Density Lipoprotein (HDL)
HDL is mainly composed of protein, lipids, cholesterols, and minor lipids. Its structure functionality includes antioxidant activity
and protective effects against pathogens (Yamamoto et al., 1990;Kassaify et al., 2005). Lipid moiety in HDL influence its electron
donating properties which can reduce the generation of reactive oxygen species (ROS). Mechanisms proposed to include the reduc-
tion of lipid hydroperoxides to non-reactive species through the electron transfer of methionine amino acid, interfering with lipid
oxidation propagation (Elias et al., 2008).
Phosvitin
Phosvitin is a highly phosphorylated molecule with 124 of 217 amino acids binds to phosphate through covalent bonds, being
serine the predominant amino acid (Byrne et al., 1984;Lei and Wu, 2012). This characteristic yield a high mineral binding capacity,
interacting with 95% of the iron present in egg yolk (Grogan and Taborsky, 1986). The addition of ascorbic acid release the phos-
vitin bonded iron, thereby increasing the lipid peroxidation in egg yolk (Nielsen et al., 2000). On the other hand, its chemical prop-
erties derive from the emulsifying functional property. When in complete protein moiety, emulsifying properties are conferred by
the interaction of the protein charged Neand Cetermini. Further, glycosylated phosvitin has shown to improve the viscoelastic
layer (Khan et al., 1999).
Livetin
Livetin is a globular water-soluble glycoprotein which makes up 30% of the total egg yolk plasma proteins. The protein is present in
three forms, namely, a-livetin, b-livetin, and g-livetin (Sugino et al., 1997b). g-livetin is primarily immunoglobulin Y (IgY) which is
an ideal substitute for mammalian IgG (Laca et al., 2015). The reported molecular weights of a-livetin, b-livetin, and g-livetin are
80,000, 45,000, and 170,000 respectively.
a-livetin has been reported to be the primary allergen responsible for the bird egg syndrome, and the protein is partially heat-
labile inhalant. The reactivity of IgE towards a-livetin was reported to reduce by almost 90% when it was heated for 30 minutes at
90 C. Only a partial cross-reactivity was observed between a-livetin and conalbumin (Quirce et al., 2001). Martin et al. (1957)
reported that although the molecular weights and tyrosine: tryptophan ratios of a-livetin and serum albumin were similar, they
were not identical proteins because of the solubility. However, newer studies reported that although there was some precipitation
of a-livetin at lower concentrations of (NH₄)₂SO₄, most of it was not precipitated by 50% saturation. Moreover, the peptide patterns
and immunological results suggested that both a-livetin and serum albumin were the same protein.
b-livetin contains 7% hexose. This protein is distinct from ovalbumin, although the molecular weights of both the proteins are
very similar. Because the immunological patterns of b-livetin were identical to serum protein, it was considered an a2-glycoprotein,
from its carbohydrate content and electrophoretic mobility. The sedimentation coefficient of 3S of b-livetin was similar to human
serum, although the sialic content was lower in b-livetin as compared to human serum proteins (Schmid and Burgi, 1961;Burgi and
Schmid, 1961).
The g-livetin or IgY is derived from IgG of hen’s serum, although it differs in from mammalian IgG in many of its chemical and
structural properties. Yolk IgY consists of Asn-linked oligosaccharides like IgG. However, the oligosaccharide composition is
different in both the immunoglobulins. The molecular weight of the heavy chains of IgY is greater than mammalian IgG
(Kovacs-Nolan and Mine, 2004). Along with compositional differences, IgG and IgY also have functional differences. For instance,
the isoelectric point of IgY is lower, and it is incapable of association with the mammalian complement, Protein G, Protein A, or
rheumatoid factors. IgY also shows lesser binding capabilities with bacterial and human Fc receptors (Kovacs-Nolan and Mine,
2004).
8Egg Proteins
Conclusion
Hen’s egg has been considered as one of the most nutritious source of food as it can sustain both life and growth. The proteins
present in the egg are nutritionally complete with a great balance of essential amino acids. Although the egg proteins are present
in all parts of the eggs, but the major concentration lies in the egg white (50%) and egg yolk (40%), while the remaining proteins
are distributed in the egg shell and egg shell membrane. Each protein component of the egg white and egg yolk is responsible for
imparting a specific physical and chemical characteristic towards the entire functionality of an egg. Moreover, environmental condi-
tions such as ionic strength, pH, and temperature treatments can modulate the functional properties of these proteins. Therefore,
a greater understanding of the egg proteins could help in the development of functional foods, as egg proteins, particularly egg white
proteins, have gained a lot of research interest in the recent years. Apart from academic research, food industries have also shown
a lot of interest in egg proteins to explore the various functional properties of eggs, which could provide them many commercial
benefits.
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Further Reading
Abdou, A.M., Kim, M., Sato, K., 2013. Functional Proteins and Peptides of Hen’s Egg Origin. InTech.
Huopalahti, R., López-Fandiño, R., Anton, M., Schade, R., 2007. Bioactive Egg Compounds. Springer-Verlag, Berlin Heidelberg.
Mine, Y., 2007. Egg Bioscience and Biotechnology. Wiley, United States of America.
Roberts, J., 2017. Achieving sustainable production of eggs. In: Safety and Quality, vol. 1. Burleigh Dodds Science Publishing.
Sim, J., Hoon, S., 2006. The Amazing Egg Nature’s Perfect Functional Food for Health Promotion, first ed. University of Alberta Hospitals, Alberta.
Stadelman, W.J., Cotterill, O.J., 1995. Egg Science and Technology, fourth ed. CRC Press, Boca Raton.
12 Egg Proteins
