Egg proteins

14 Egg proteins M. Anton, INRA Nantes Unite 1268 BiopolymeÁres Interactions Assemblages, France and F. Nau and V. Lechevalier, UMR INRA Science et Technologie du Lait et de l'Oeuf, France

Abstract: This chapter deals with the chemical composition and structural characteristics of egg yolk and white in relation to three important functional properties: emulsifying, foaming and gelling properties. Key words: egg, yolk, white, emulsions, foams, gels, structure, assemblies, interfaces.

14.1

Introduction: technofunctional uses of egg constituents

Hen egg was categorised by Baldwin in 1986 as a polyfunctional ingredient, as it can simultaneously realise several technological functions in the same formulated foodstuff. Its emulsifying, foaming, gelling, thickening, colouring and aromatic properties make it still today a universal basic ingredient for the domestic kitchen and the food processing industry. Whereas egg yolk is well recognised for its emulsifying properties, egg white (or albumen) is a reference in terms of foaming and both parts are used as gelling ingredient in many foods. Yolk takes part in the formation and the stabilisation of emulsions. In spite of the intensive use of yolk in formulated foodstuffs, and since the invention of mayonnaise three centuries ago, the role of its major constituents is not clear because of its complex structure. Yolk is a mixture of proteins and lipids forming natural assemblies at various scales. These natural assemblies contribute to the nano- and the microstructure of yolk. Thus, an understanding of the emulsifying properties of yolk lies in the comprehension of these various levels of structure.

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The exceptional foaming properties of the albumen are also the base of traditional recipes among which meringues act certainly as reference. Indeed, the extreme simplicity of their formula (albumen and sugar, possibly added with flavours) allows albumen to express in an optimal way its foaming properties. However, the technological parameters influence the final quality of foam obtained, and three types of meringues (traditional meringue, Swiss meringue and Italian meringue) can be distinguished, depending on whether whipping is achieved in the presence or absence of sugar, and at ambient or warm temperature. But there is also a great number of other products in which previously foamed albumen is added, which are either fat-free formulas (angel food cake) or lipid-containing formulas (spoon biscuits, `sponge' cake, blown). In such products, the complexity of the phenomena is extreme, foaming and emulsification taking place simultaneously, which makes the control of the physico-chemical and technological parameters of these operations very delicate. Concerning the gelling properties of albumen and yolk, they are related to the heat-gelation capacity of egg proteins. Then, these properties imply a cooking step during the food processing. The heat gelation of egg proteins completely conforms to the model of heat gelation of globular proteins. The corresponding mechanisms have been extensively studied, on egg proteins as well as on other ones, and the key technological parameters have now been identified. However, the addition of other ingredients in mixture with egg (polysaccharides, for example) complicates the understanding of the egg gelation behaviour, and developments with more complex models are still needed.

14.2

Physico-chemistry and structure of egg constituents

14.2.1 Egg yolk Chemical composition Yolk correspond to 36% of whole hen egg weight. Its dry matter is about 50± 52% according to the age of the laying hen and the duration of preservation (Kiosseoglou, 1989; Thapon and Bourgeois, 1994; Li-Chan et al., 1995). The compositions of fresh and dry yolks are presented in Table 14.1: the main components are lipids (about 65% of the dry matter) and the lipid to protein ratio is about 2:1. Yolk lipids are exclusively associated with lipoprotein assemblies. They are made up of 62% triglycerides, 33% phospholipids, and less than 5% cholesterol. Carotenoids represent less than 1% of yolk lipids, and give it its colour. Proteins are present as free proteins or apoproteins (included in lipoprotein assemblies). The interactions between lipids and proteins result in the formation of lipoproteins (low and high density), which represent the main constituents of yolk. Macrostructure and main constituents Yolk is a complex system with different structuration levels consisting in aggregates (granules) in suspension in a clear yellow fluid (plasma) that contains

Egg proteins 361 Table 14.1

Composition of hen egg yolk

Water Lipids Proteins Carbohydrates Minerals

Fresh yolk (%)

Dry yolk (%)

51.1 3.6 16.0 0.6 1.7

Ð 62.5 33.0 1.2 3.5

Source: Powrie and Nakai (1986)

lipoproteins and proteins. Granules consist in circular complexes ranging in diameter from 0.3 m to 2 m (Chang et al., 1977). Consequently, yolk can be easily separated into two fractions after a dilution (two times) with 0.3 M NaCl and a centrifugation at 10,000 g (30 min) according to the method of McBee and Cotterill (1979): a dark orange supernatant called plasma and a pale pellet called granules (Fig. 14.1). Granules represent 22% of yolk dry matter, accounting for about 50% of yolk proteins and 7% of yolk lipids. The dry matter content of granules is about 44%, with about 64% proteins, 31% lipids and 5% ash (Dyer-Hurdon and Nnanna,

Fig. 14.1 Fractionation of plasma and granules from hen egg yolk.

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Table 14.2

Repartition of hen egg yolk constituents Yolk D.M. (%)

Yolk lipids (%)

Yolk proteins (%)

Lipids (%)

Proteins (%)

100

100

100

64

32

Plasma LDL Livetins Others

78 66 10 2

93 61 Ð Ð

53 22 30 1

73 88 Ð Ð

25 10 96 90

Granules HDL Phosvitin LDLg

22 16 4 2

7 6 Ð 1

47 35 11 1

31 24 Ð 88

64 75 95 10

Yolk

Source: Powrie and Nakai (1986)

1993; Anton and Gandemer, 1997). They are mainly constituted by high density lipoproteins (HDL) (70%) and phosvitin (16%) linked by phosphocalcic bridges between the phosphate groups of their phosphoseryl residues (Burley and Cook, 1961; Saari et al., 1964). Low density lipoproteins (LDL) (12%) are included in the granular structure (Table 14.2). At low ionic strength, granules mainly form insoluble HDL-phosvitin complexes linked by phosphocalcic bridges as HDL and phosvitin contain a high proportion of phosphoserin amino acids able to bind calcium (Causeret et al., 1991). The numerous phosphocalcic bridges make the granule structure very compact, poorly hydrated, weakly accessible to enzymes, and lead to an efficient protection against thermal denaturation and heat gelation. At an ionic strength over 0.3 M NaCl, the phosphocalcic bridges are disrupted because monovalent sodium replaces divalent calcium. In such conditions, the solubility of granules reaches 80% because phosvitin is a soluble protein and HDL behave like soluble proteins (Cook and Martin, 1969; Anton and Gandemer, 1997). Complete disruption of granules occurs when ionic strength reaches 1.71 M NaCl. Acidification or alkalinisation similarly cause the disruption of granules and the solubilisation of these constituents by increasing the number of the positive (NH3+) or negative (COO-) charges inducing electrostatic repulsions between granule constituents. Recently, we have established (Sirvente, 2007) a phase diagram drawing the different states of granules as a function of pH and ionic strength (Fig. 14.2). Plasma comprises 78% of yolk dry matter and is composed of 85% LDL and 15% livetins (Burley and Cook, 1961; Table 14.2). It forms the aqueous phase where yolk particles are in suspension. It accounts for about 90% of yolk lipids (including nearly all the carotenoids), and 50% of yolk proteins. Plasma contains about 73% lipids, 25% proteins and 2% ash. Lipids of plasma are distributed thus: 70% triglycerides, 25% phospholipids and 5% cholesterol.

Egg proteins 363

Fig. 14.2 Physical state of granules as function of pH and ionic strength.

LDL are spherical particles (17±60 nm in diameter with a mean of about 35 nm) with a lipid core in a liquid state (triglycerides and cholesterol esters) surrounded by a monofilm of phospholipid and protein (Cook and Martin, 1969; Evans et al., 1973). LDL are soluble in aqueous solution (whatever the pH and ionic conditions) due to their low density (0.982). Phospholipids take an essential part in the stability of the LDL structure because association forces are essentially hydrophobic (Burley, 1975). Some cholesterol is included in the phospholipid film, increasing its rigidity. LDL are composed of 11±17% protein and 83±89% lipid, out of which 74% is neutral lipid and 26% phospholipid (Martin et al., 1964). 14.2.2 Egg white Egg white represents about 60% of the total egg weight. It consists of an aqueous protein solution, containing few minerals and carbohydrates (Table

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Handbook of hydrocolloids Table 14.3 Composition of hen egg white % of hen egg white Water Lipids Proteins Carbohydrates Minerals

88.0 Ð 10.6 0.8 0.6

Source: Thapon and Bourgeois (1994)

14.3). During egg storage, different physico-chemical modifications happen, among them the CO2 departure that induces a pH increase, from 7.5 at the laying moment to 9.5 after a few days. This pH modification should be the cause of the egg white liquefaction, because of the dissociation of a protein complex (ovomucin-lysozyme complex) (Kato et al., 1975). Another evolution observed concerns the ovalbumin modification toward S-ovalbumin, which is a more heat-stable form (Smith and Back, 1965), resulting from isomerisation of three serine residues (Yamasaki et al., 2003). Proteins Proteins represent more than 90% of the dry matter of egg white, but until very recently, only the major ones have been identified. However, the recent and powerful techniques for separation and analysis enabled the identification of many minor proteins (Table 14.4) (GueÂrin-Dubiard et al., 2006; Mann, 2007). The egg white proteins are predominantly globular proteins, and acidic or neutral, except lysozyme and avidin which are highly alkaline proteins. All are glycosylated, except cystatin and the major form of lysozyme. Some of them are very heat-sensitive and/or sensitive to surface denaturation, explaining their noteworthy functional properties. The major egg white protein (more than 50% of the total proteins) is ovalbumin, a 45 kDa globular and phosphorylated protein. Half of its amino acids are hydrophobic, and one-third are electrically charged, essentially negatively at physiologic pH. Ovalbumin possess six buried Cys residues, two being involved in a disulfide bridge (Cys73-Cys120). Ovalbumin is then the only egg white protein with free thiol groups, capable of inducing some rearrangements with variations of storage conditions, pH and surface denaturation. Ovotransferrin (13% of total proteins) molecular weight is around 78 kDa. This protein consists of two lobes, each containing a specific binding site for iron (or copper, zinc, aluminium) (Kurakawa et al., 1995). It is the most heatsensitive egg white protein, but the complexation of iron or aluminium significantly increases its heat stability (Lin et al., 1994). OvomucoõÈde is a highly glycosylated protein (up to 25% carbohydrates, w/w) of 28 kDa. At pH 7, its denaturation temperature is around 77 ëC, but this protein

Egg proteins 365 Table 14.4 Composition and some physico-chemical and functional properties of egg white proteins %

Mw (kDa)

pI

Ovalbumin Ovalbumin Y Ovalbumin X Ovotransferrin OvomucoõÈd Ovomucin

54 5 0.5 13 11 1.5±3.5

45 44 56 76 28 230±8300

5 5.2 6.5 6.7 4.8 4.5±5

Lysozyme Ovoinhibitor Ovoglycoprotein Flavoprotein Ovostatin Cystatin Avidin Ex-FABP Cal gamma TENP

3.5 0.1±1.5 0.5±1 0.8 0.5 0.05 0.05 nd nd nd

14.4 49 24.4 32 760±900 12.7 68.3 18 20.8 47.4

10.7 5.1 3.9 4 4.6 5.1 10 5.5 6 5.6

nd

18

6.4

Protein

Hep 21

Major biological properties Immunogenic phosphoproteine nd nd Iron binding, bacteriostatic activity Trypsin inhibitor Highly glycosylated, viral hemaglutination inhibition Lysis of Gram‡ bacteria wall Serine protease inhibitor nd Riboflavin (vitamin B2) binding Serine protease inhibitor Cysteine protease inhibitor Biotine binding Lipocaline family Lipocaline family BPI (bactericidal permeabilityincreasing protein) family uPar/Ly6/Snake neurotoxin family

Sources: Li-Chan and Nakai (1989), Stevens (1991), GueÂrin et al. (2006).

is much more heat resistant at acidic pH (Lineweaver and Murray, 1947). Ovomucin is also a highly glycosylated protein, with a very high molecular weight (104 kDa). Electrostatic interactions can be observed between ovomucin and some of the other egg white proteins. In the freshly laid eggs (pH 7.5), the carboxylic groups of the ovomucin sialic acids especially interacts with the NH3+ of lysozyme lysine residues to form a lysozyme-ovomucin complex that may be responsible for the gel-like structure of egg white (Kato et al., 1975). Lysozyme is a small (14 kDa) globular, and strongly basic protein. Its structure is very rigid, stabilised by four disulfide bridges. Glucidic and mineral fractions The glucidic fraction of egg white consists of free glucose (0.5% w/w) and carbohydrates linked to proteins (0.5% w/w). The mineral fraction is predominantly composed of Na+, K+ and Clÿ, as free minerals, whereas P and S are essentially constitutive elements of proteins. Egg white also contains CO2, in equilibrium with bicarbonate, which plays a major role for pH control (Thapon, 1994).

14.3

Egg yolk emulsions

14.3.1 Basic principles Emulsifying activity is related to the capacity of surface active molecules to cover the oil±water interface created by mechanical homogenisation, thus

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reducing the interfacial tension. Consequently, the more active the emulsifying agent, the more the interfacial tension is lowered. Emulsion stability indicates the capacity to avoid flocculation, creaming, and/or coalescence of oil droplets. Creaming and flocculation are reversible phenomena which can be avoided by a simple agitation of the emulsion. Coalescence is the irreversible fusion of oil droplets due to the rupture of the interfacial film created by emulsifying agents. This phenomenon leads to a complete destruction of the emulsion. This relates the importance of the structure and the viscoelasticity of the interfacial film. 14.3.2 Role of egg yolk constituents In researching the principal contributor to yolk emulsifying properties, numerous authors have separated yolk into its main fractions: plasma and granules. Large similarities have been observed between emulsifying properties of yolk and plasma, whereas emulsions made with granules behaved very differently (Dyer-Hurdon and Nnanna, 1993; Anton and Gandemer, 1997; Le Denmat et al., 2000). Specifically, emulsions made with granules are more coarse (more important oil droplet size) than emulsions made with yolk and plasma, and notably at acidic pH where granules are not soluble (Le Denmat et al., 2000) (Fig. 14.3). Concerning the parameters of emulsion stability (creaming), we showed (Le Denmat et al., 2000) that emulsions made with yolk and plasma had the same creaming rate, in function of the medium conditions, whereas emulsions made with granules behaved very differently (Fig. 14.3). Consequently, these studies demonstrated that yolk emulsifying power was situated in plasma. Among plasma constituents, some authors demonstrated that LDL are better emulsifiers than bovine serum albumin (BSA) (Mizutani and Nakamura, 1984) and casein (Shenton, 1979). Even though some authors suggested that, in certain conditions, HDL were more efficient than LDL to form and stabilise O/W emulsions (Hatta et al., 1997; Mine, 1998), a large number of studies confirm the prevalent role of LDL in yolk emulsions. These findings have been confirmed recently (Aluko et al., 1998; Mine and Keeratiurai, 2000; Anton et al., 2003; Martinet et al., 2003). In particular, it has been established that LDL made emulsions finer than HDL, along different conditions of pH and ionic strength (Martinet et al., 2003). The next question is how to explain the exceptional efficiency of LDL at the interfaces. 14.3.3 Importance of assemblies Given that any destructurating treatment affects the emulsifying properties of LDL, it appears that the integrity of the structure of LDL seems essential to ensure their interfacial properties (Tsutsui, 1988). Direct adsorption of apoproteins and phospholipids from LDL is not easy because of the nonsolubility of these species in water or in aqueous buffer. So the interactions between apoproteins and lipids to assemble the LDL particles are essential to

Egg proteins 367

Fig. 14.3 Mean droplet diameter (d3.2) and creaming index (Icr) in oil/water emulsions (30 : 70) prepared with yolk, plasma and granules, protein concentration: 25 mg/ml, homogenisation pressure: 200 bars, n ˆ 3.

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Fig. 14.4 /A isotherms of the different lipid constituents extracted from LDL and spread at the air±water interface; neutral lipids = 85 g, phospholipids = 198 g, total lipids = 287 g, compression rate = 100 cm2/min.

transport the surfactants in a soluble form in the neighbourhood of the interface and then to release them at the interface. Using Langmuir film balance (air±water interface), three phase transitions have been detected in compression isotherms and these three transitions (19, 41 and 54 mN/m) have been attributed, respectively, to neutral lipids, apoproteins and phospholipids by comparison with films of neutral lipids, phospholipids and total lipids extracted from LDL (Fig. 14.4) (Martinet et al., 2003). The transition observed at 19 mN/m corresponds to the collapse of neutral lipids, and the transition at 54 mN/m corresponds to phospholipid collapse. These different transitions show that LDL actually break down when they come into contact with the interface to release neutral lipids, phospholipids and apoproteins from the lipoprotein core and to allow their spreading. In a recent study made with atomic force microscopy (AFM) after a Langmuir±Blodgett transfer of the layers from the air±water interface to a silica plate, it has been shown that the second transition (previously attributed to apoproteins alone) is not due to apoproteins alone, but to apoprotein±lipids complexes (Dauphas et al., 2006). So, it has been deduced that LDL serve as vectors of surfactant constituents (apoproteins and phospholipids) that could not be soluble in water, until the interface. At this step the conservation of the LDL structure is essential. Once LDL are near the interface, the structure is then broken up to release surfactant constituents at the interface (Fig. 14.5). Furthermore, comparing interfacial behaviour of LDL and liposomes (double phospholipid layer not containing proteins), it has been shown that the apoproteins situated on the LDL surface start the LDL disruption mechanism by

Egg proteins 369

Fig. 14.5 Hypothetical mechanism of LDL adsorption at an oil±water interface as compared with liposome behaviour.

their initial anchorage. This anchorage provokes an unfolding of the protein leading to the destabilisation of the external layer of the LDL. Then this phenomenon could be followed by a deformation of the particle due to the creation of a neutral lipid lens conducive to the spreading of the LDL constituents. In the case of liposomes, without external proteins, the structure remains steady at the interface and then this structure is not able to adsorb efficiently and to decrease interfacial tension (Fig. 14.5).

14.4

Egg white foams

14.4.1 Formation and stabilisation mechanisms Foam formation is a highly energetic and dynamic process, in which interfacial area is created. The ability of a protein solution, such as egg white, to foam depends on protein structure and conformation, depending themselves on extrinsic factors such as pH, ionic strength, etc. The formation mechanism of globular protein foams can be divided into three phases happening near gas bubbles: protein diffusion towards the air±solution interface, conformation changes of adsorbed proteins, and irreversible rearrangement of the protein film (McRitchie, 1991). Foams are short-lived states and there is any correlation between foam stability and protein adsorption kinetic (Dickinson, 1996). Foam stability, indeed, depends on protein association at the air±solution interface to form a

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continuous intermolecular network. Foam stability is affected by the protein film cohesion, drainage and Ostwald disproportionation. 14.4.2 Interfacial properties of egg white proteins Interfacial properties of egg white proteins are responsible for egg white's excellent foaming properties. Table 14.5 gathers some data on the kinetics of diffusion towards the air±solution interface of three major egg white proteins. Ovalbumin interfacial behaviour is well known, since a large set of data is available about its tensioactivity, adsorption kinetics, interfacial shear and dilatational rheology (de Feijter et al., 1978; de Feijter and Benjamins, 1987; Benjamins and van Voorst Vader, 1992; Benjamins and Lucassen-Reynders, 1998; Damodaran et al., 1998; Lucassen-Reynders and Benjamins, 1999; Pezennec et al.; 2000; Razumovsky and Damodaran, 2001; Croguennec et al., 2007) but also on its structure at the air±water interface (Renault et al., 2002; Lechevalier et al., 2003, 2005; Kudryashova et al., 2003). It is now known that ovalbumin forms a single layer at the air±water interface, whatever its concentration in the bulk (Renault et al., 2002). As for ovalbumin, lysozyme interfacial behaviour has been extensively studied (de Feijter and Benjamins, 1987; Damodaran et al., 1998; Razumovsky and Damodaran, 2001; Kim et al., 2002; Postel et al., 2003; Chang et al., 2005; Roberts et al., 2005; Perriman and White, 2006) as well as its structure at the air±water interface (Lechevalier et al., 2003, 2005). However, its interfacial behaviour differs as lysozyme forms films that are much thicker than a protein monolayer whereas the surface pressure is definitely smaller than the ovalbumin one (Le Floch-FoueÂre et al., 2009). These different behaviours observed on planed air±water interface result in different foaming properties. Ovalbumin foaming properties are much better than those of lysozyme, since in native state at pH 7.0, the foaming capacity of lysozyme is very weak (Townsend and Nakai, 1983), probably because of its little surface hydrophobicity and its rigidity due to its four disulfide bonds. Egg white proteins thus show different behaviour at 2D and 3D air±water interfaces. When they are in mixture, their behaviour is again different. Indeed, Damodaran et al. (1998) showed that the adsorption kinetics of egg white proteins are different depending on whether they are in single protein systems or in mixture. They suggested the formation of electrostatic complexes between positively charged lysozyme and other negatively charged egg white proteins. Moreover, the mixture ovalbumin-lysozyme forms films that are much thicker than those of both proteins in single protein systems, suggesting a synergy in interfacial adsorption between the two proteins (Le Floch-FoueÂre et al., 2007). 14.4.3 Egg white foams Egg white is the reference for foaming properties: compared with other protein ingredient of vegetable or animal origin, it still offers the best foaming properties (Vani and Zayas, 1995; Matringe et al., 1999; Pernell et al., 2002;

Table 14.5 Parameters of the kinetic of diffusion towards the air±solution interface of three major egg white proteins Parameters

Ovalbumin

Apparent diffusion coefficient (10ÿ10 m2 sÿ1)

0.5 (C=10ÿ4% prot.) (in solution: 0.7)

Surface concentration (mg mÿ2)

Surface pressure (mN mÿ1)

Lag phase

Ovotransferrine

0.5 to 1 (C=0.1% prot.) 1.6 (C=10ÿ4% prot.) 1.5 (C=5.4
10ÿ4% prot.) 2.1 (native protein) to 2.9 (heat-treated protein) (C=0.01% prot.)

0.8 (C=1.2
10ÿ4% prot.)

1 (C=10ÿ4% prot.) 14 (C=5.4
10ÿ4% prot.) 24 (C=0.01% prot.)

YES if C<0.01% Not enough molecules at the interface to create an increase of  NO

2.5 (C=1.2
10ÿ4% prot.)

Lysozyme

Reference

0.2 (C=10ÿ4% prot.) (in solution: 1) 0.15 (C=1.5
10ÿ4% prot.)

De Feijter and Benjamins (1987) Xu and Damodaran (1993) Pezennec et al. (2000)

2.4 (C=10ÿ4% prot.)

De Feijter and Benjamins (1987) Damodaran et al. (1998) Pezennec et al. (2000) Croguennec et al. (2007)

0.5 (C=0.35
10ÿ4% prot.)

1.4 (pH 4) to 3 (pH 11) (C=0.1% prot.)

Perriman and White (2006)

3.5 (C=10ÿ4% prot.)

De Feijter and Benjamins (1987) Damodaran et al. (1998) Pezennec et al. (2000) Roberts et al. (2005)

2.5 (C=0.35
10ÿ4% prot.) 8 (pH 5.6) to 14 (pH 11) (C=0.012% prot.) 9 (C=5
10ÿ4% prot.) to 24.5 (C=0.1% prot.)

YES

YES if C<0.01% Not enough molecules at the interface to create an increase of  YES

Chang et al. (2005) De Feijter and Benjamins (1987) Damodaran et al. (1998)

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Table 14.6

Interfacial characteristics of the main egg white proteins

Protein

Surface tension (mN mÿ1)

Globulins Ovalbumin Ovotransferrin Lysozyme OvomucoõÈd Ovomucin Mixture in the egg white ratio

45.4 51.8 42.4 42.0 39.0 nd 46.7

Foamability (cm3 gÿ1 minÿ1) 4.71 0.59 0.34 0.12 0 0 3.08

Source: Mine (1995)

Foegeding et al., 2006; Davis and Foegeding, 2007). Egg white can be considered as a solution of efficient surfactants. Its proteins are amphiphilic and show a relatively high surface hydrophobicity, thus diffusing quite rapidly towards the air±water interface where they adsorb efficiently. Their molecular flexibility ensures conformational rearrangement at the interface, resulting in a great decrease in surface tension. Their ability to form a continuous intermolecular network, especially when a certain denaturation level is previously obtained, enable them to form a viscoelastic interfacial film responsible for foam stability. However, egg white proteins do not present at the same level these different characteristics, and thus do not participate in the same way to egg white foaming properties (Table 14.6). A lot of studies have been performed on the different egg white protein foaming properties (Nakamura, 1963; Johnson and Zabik, 1981a; Mine, 1995). However, it is now well known that the complexity and the synergy of the phenomena mean that it is impractical to distinguish the role of the different egg white properties (Lechevalier et al., 2005). It is thus quite difficult to predict foaming properties of any mixture of egg white proteins since phenomena of competition for the interface and possible exchange between proteins at the air±water interface may occur. Nevertheless, foaming properties of isolated egg white proteins are always lower than those of egg white, which tend to confirm the existence and the role of the interactions between proteins. It is thus generally admitted that the natural coexistence in egg white of alkaline protein (lysozyme) and acid ones (most of the other) enables electrostatic interactions, thus explaining the good foaming properties of egg white (Poole et al., 1984; Damodaran et al., 1998). Egg white foams are an integral component of many foods such as meringue, nougat and angel food cake. A recent study showed that the properties of foams do not predict performance in angel food cake (Foegeding et al., 2006). 14.4.4 Key parameters Many physico-chemical parameters are susceptible to influence foam formation and stability. In the specific case of egg white proteins, surface hydrophobicity (that conditions the efficiency of protein adsorption at the air±water interface),

Egg proteins 373 the number of disulfide bonds (that conditions protein flexibility/rigidity) and the number of free sulfhydryl groups (that conditions protein reactivity) are decisive in the structural modifications that occur at the air±water interface. Moreover, the number and the nature of inter- and intramolecular interactions determine the rheological properties of the interfacial film and so foam stability. These interactions are favoured by a certain degree of denaturation, however, too much denaturation weakens the interfacial film and the foam collapses (Kinsella, 1976; Trziszka, 1993; Kato et al., 1994; Van der Plancken et al., 2007). Another special feature of egg white foams is their dependence on thick egg white proportion and quality. Foamability increases with egg white natural liquefaction during its storage (Sauveur et al., 1979; Thapon, 1981; Baldwin, 1986), whereas foam stability decreases (Nau et al., 1996). Egg white foaming properties can also be improved by the addition of sucrose (effect on foam stability) and sodium chloride (effect on foamability), as suggested by Raikos et al. (2007a).

14.5

Gels

14.5.1 Basic principles A gel consists of polymers linked through covalent and/or non-covalent interactions, to create a three-dimensional network. In whole egg as well as in white and yolk, proteins are responsible for the gelling properties. Gelation occurs when the protein stability in solution is modified, i.e., when the equilibrium between attractive (Van der Waals) and repulsive (electrostatic, steric) interactions is disrupted. The electrostatic repulsions vary with the net charge of the proteins, that means with the ionisable protein groups and with the physico-chemical characteristics of the solvent (pH, ionic strength). The treatments that decrease the repulsive interactions, such as adjustment of pH at proteins pI or addition of salts, induce destabilisation and thus can result in the formation of aggregates or gels. Moreover, some treatments can modify the protein structure, with consequences for the repulsive and attractive interactions mentioned above. This is especially the case during heat treatments, which are the major technological treatments used in the food industry for egg white and yolk gelation. Heat-induced gelation of egg conforms completely with the model of heat gelation of globular proteins. It is a two-step phenomenon: in the first stage, unfolding of native proteins occurs, disrupting the well-defined secondary and tertiary structures and producing denatured proteins exposing their inner hydrophobic regions; following unfolding, the denatured proteins interact to form high molecular weight aggregates that can further interact with each other to result in a three-dimensional gel (Clark et al., 2001). The unfolding and aggregation steps depend on many factors (protein concentration, ionic strength, pH, presence of sucrose, etc.) that can modify the number and/or the kind of interactions, with final consequences on the gel rheology. In the heat-induced

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gels of egg proteins, the interactions involved are predominantly hydrophobic and electrostatic, but some highly energetic interactions can be observed (disulfide bridges); thiol and amine groups are indeed very reactive, especially in alkaline conditions. 14.5.2 Egg yolk gels Yolk undergoes a gelation when it is subjected to a freezing-thawing process or a heat treatment. LDL are responsible for yolk gelation, while the other constituents of yolk do not participate directly (Kojima and Nakamura, 1985; Kurisaki et al., 1981; Nakamura et al., 1982; Tsustui, 1988; Wakamatu et al., 1982; Le Denmat et al., 1999). Freezing-thawing gelation of yolk appears for a temperature below ÿ6 ëC (Lopez et al., 1954). This gelation is undesirable because it makes yolk difficult to handle. Freezing-thawing gelation is influenced by rate and temperature of freezing and thawing, and length and temperature of storage (Kiosseoglou, 1989). Rapid freezing and thawing results in less gelation than slow freezing and thawing. Freezing-thawing gelation is partially reversible and the initial viscosity of yolk is recovered after heating for 1 h at 45±55 ëC. The mechanism of freezing-thawing gelation of LDL remains hypothetical. Freezing causes the formation of ice crystals which mobilise water (at ÿ6 ëC, about 80% of the water in yolk is in ice crystals) and causes a dehydration of apoproteins of LDL. This dehydration favours a rearrangement of apoproteins of LDL, interactions between their amino acid residues and an aggregation which leads to gelation. LDL is the constituent of yolk responsible for heat-induced gelation of yolk (Saari et al., 1964). LDL solution (4% w/v) start denaturing at 70 ëC and form gels at 75 ëC (Tsutsui, 1988). LDL solutions, heated at 80 ëC for 5 min, form more stable gels than ovalbumin and BSA (Kojima and Nakamura, 1985). Unlike ovalbumin and BSA, LDL present a heat-induced gelation in a large range of pH (4±9) with a minimal value around their pHi (Nakamura et al., 1982). Between pH 6 and 9, LDL solutions form coagulum gels (opaque) whereas LDL solutions form translucents gels for extreme pH (4±6 and 8±9) (Kojima and Nakamura, 1985; Nakamura et al., 1982). Heat-induced gelation of yolk is governed by the unfolding of proteins during heating. Then the functional groups are exposed and attracted to one another through hydrophobic bonds resulting in a gel (Nakamura et al., 1982). The primary stage of the two phenomena (freezing-thawing and heat-induced gelation) is the disruption of the LDL structure (Kurisaki et al., 1981). This disruption is favoured by dehydration in the case of freezing-thawing, or by unfolding under heating. Lipid-protein interactions are disrupted under freezing or heating and interactions between proteins are increased. These interactions are both principally of non-polar nature because a LDL gel is solubilised by SDS which interacts with the hydrophobic residues of apoproteins (Mahadevan et al., 1969). The aggregation product of LDL certainly contains lipids included in the structure

Egg proteins 375 (Tsutsui, 1988). Apoproteins of LDL present a large proportion of hydrophobic amino acids and, consequently, they have a high ability to form such gels. More recently, Le Denmat et al. (2000) have measured the critical concentrations (Cg) for heat gelation of dispersions of yolk, plasma and granules in pH and NaCl ranges of respectively 3±7 and 0.15±0.55 M. In all cases, the domain Cg for plasma is 12±28 mg protein/ml, whereas it is 26±120 mg protein/ml for granules. For yolk solution Cg is comprised between 16 and 39 mg protein/ml. This confirms the preponderant influence of LDL, the major compound of plasma, in the heat gelation of yolk. This underlines the excellent capacity of granules to resist to heat treatments, that could be used for industrial applications. 14.5.3 Egg white gels The heat gelation of egg white is used in many food applications involving a cooking step. Except ovomucin and ovomucoõÈd, all the egg white proteins coagulate when heated (Johnson and Zabik, 1981b). But the heat sensitivity of egg white proteins varies significantly: the temperature of denaturation at pH 7 in egg white is 84.5, 74 and 65 ëC for ovalbumin, lysozyme and ovotransferrin, respectively (Donovan et al., 1975). Ovotransferrin is then the more heatsensitive, which is why it is generally considered as the gelation initiator, and finally as a limiting factor considering the gelling properties. Therefore, ovotransferrin elimination has been suggested to improve egg white gelling properties (Kusama et al., 1990). However, ovotransferrin is more stable at alkaline pH, at high ionic strength, and when metal ions are bound on it. Thus, the gelation temperature of egg white can also be significantly increased by modification of these parameters, and especially by Fe3+ or Al3+ addition (Cunningham and Lineweaver, 1965). The extent and the kind of the interactions between the denatured proteins depend on the protein structure, that means on the extent of unfolding at the end of the denaturation step. Indeed, the unfolding governs the more or less important exposure of reactive groups or regions on the protein molecule. The interactions also depend on the physico-chemical conditions that can be either limiting or favouring, resulting in an increase or a decrease of aggregation rate respectively, and then in a decrease or an increase of the denaturation extent before interactions take place (Totosaus et al., 2002). These mechanisms have been extensively studied for egg white and ovalbumin heat-gelation, with a focus on ionic strength effect on the structure and characteristics of the gels (Holt et al., 1984; Woodward and Cotterill, 1986; Woodward, 1990; Croguennec et al., 2002; Raikos et al., 2007b). Heat denaturation induces an increase of the protein surface hydropbobicity. When heating occurs at high ionic strength, the protein charges are screened, inducing a shielding effect on the repulsive forces between proteins, thus favouring the hydrophobic interactions (Doi, 1993). In these conditions, random aggregates of slightly denatured proteins appear, corresponding to opaque gels, with low rigidity, elasticity and water retention capacity. On the other hand, at low ionic

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strength, the electrostatic repulsions are so high that it delays the aggregation (Raikos et al., 2007b), favouring denaturation. Finally, the further aggregation involves some specific area (hydrophobic regions), and induces linear polymeric aggregates. Once placed in higher ionic strength conditions, these aggregates can interact to form performing gels. Thus, a two-step heating process has been proposed to produce translucent gels from egg white, very firm and elastic, and with an exceptional water retention capacity (Kitabatake et al., 1988a). As well as ionic strength influences the net charge of the proteins, pH is another major parameter for egg white gelation control. Close to their pI, the proteins tend to form random aggregates, similar to those obtained at high ionic strength. This phenomenon explains the minimal rheological properties of the egg white gels around pH 5. In contrast, at alkaline pH, egg white offers the best gelling properties (Ma and Holme, 1982; Kitabatake et al., 1988b). The higher reactivity of the thiol groups in these pH conditions probably also contributes to the improvement of the gelling properties, because of disulfide bridges taking place. On the other hand, at acidic pH (2.0), the limited protein solubility would be responsible for the low gelation temperature and the low rheological properties observed (Raikos et al., 2007b). To improve the gelling properties of egg white, Kato et al. (1989) proposed an original approach consisting of an extensive denaturation of proteins while preventing aggregation. Such conditions can be obtained by heating of egg white powder at high temperatures (80 ëC) for a long time (up to 10 days). This treatment increase protein flexibility and exposure of reactive groups that can further interact to strengthen the gel formed when previously dry-heated egg white is solubilised and heated in solution. The efficiency of this process can be improved by pH control (Mine, 1996, 1997). The dry-heating process is today the basis for mass production practices of high-gel egg white powders.

14.6

Conclusion

Hen egg contains very high functional proteins, lipids and lipoproteins. These functionalities are due partly to their chemical composition and structure, and partly to the supramolecular assemblies they form naturally or under the action of thermo-mechanical treatments during industrial processes. One of the major challenges for the future is the control of the design of these assemblies and the understanding of their functionalities (interfacial, emulsifying, foaming, phase separation, etc.) to enhance the quality of existing products or to conceive innovative products.

14.7

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