modification of functional properties of egg-white proteins

Review

Modification of functional properties of egg-white proteins

Lydia Campbell, Vassilios Raikos and Stephen R. Euston

Contents1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3692 Heat-induced structural changes of globular proteins 3692.1 Denaturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3702.2 Measurement of the denaturation degree . . . . . . . . . . 3703 Structural and functional properties of egg-white

protein systems as a result of heat treatment . . . . . . . 3713.1 Solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3713.2 Gelation/coagulation . . . . . . . . . . . . . . . . . . . . . . . . . . 3713.3 Foaming ability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3713.4 Effect of spray drying . . . . . . . . . . . . . . . . . . . . . . . . . 3724 Functional properties of egg-white heated in the dry

state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3724.1 Solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3724.2 Heat-induced gelling ability . . . . . . . . . . . . . . . . . . . . 3724.3 Emulsifying ability . . . . . . . . . . . . . . . . . . . . . . . . . . . 3725 Combined effects of sugar and salt on structural and

functional properties of heat-treated egg-white proteins:a novel approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373

5.1 Heat stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3735.2 Egg-white properties after spray-drying . . . . . . . . . . . 3736 The Maillard reaction: basis for improved functional

properties of egg-white treated as stated in the novelapproach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373

6.1 Effect of heating in the presence of sugars on globularprotein structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374

6.2 Impact of Maillard reaction on the functional propertiesof egg-white proteins . . . . . . . . . . . . . . . . . . . . . . . . . . 374

6.3 Solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3746.4 Gelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3756.5 Emulsifying properties . . . . . . . . . . . . . . . . . . . . . . . . 3756.6 Foaming ability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3757 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3768 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376

1 IntroductionThe food manufacturer has a duty to ensure safe food prod-

ucts that are free from pathogenic microbial contamination.Because of this, foodstuffs are often subject to intense heattreatment designed to kill microbial contamination, or are for-mulated at low pH where, among other factors, microbialgrowth is prevented. This can present problems in foods whereproteins play a major role in the food structure. Proteins areused as emulsifiers in many formulated fat-containing foods.That is they are used to encapsulate fat in the form of smalldroplets thereby stabilising it against separation. During foodprocessing, problems may arise when emulsified products aresubject to high heat or low pH, as many food proteins are labileto one or both of these conditions. Upon pasteurisation ofwhole egg, denaturation of egg albumen could occur, whichwould result in its precipitation upon acidification (acidifica-tion is a necessary step in mayonnaise manufacture), therebyresulting in decreased emulsion stability. Conventionally there-fore, manufacturers in the egg processing industry have goneto considerable lengths to avoid any denaturation of the albu-men as far as possible in order to prevent the albumin fromcoagulation. There would be a significant benefit to the foodmanufacturer if they had available heat-stable and acid-stablefood protein such as whole egg or egg-white, for use as emulsi-fiers and thickeners.

Whole egg is a complex mixture of proteins, water, carbohy-drate, fat, ash and cholesterol. The main components of wholeegg are egg-white (albumin) and egg-yolk. On a weight basis,egg-white (58%) is about twice as much as egg-yolk (31%),whereas the shell contributes about 11% to the total egg-weight. Egg-white contains about 88% of water, 11% of pro-teins, 0.2% of fat and 0.8% of ash [1]. The most abundant pro-teins in egg-white are ovalbumin, conalbumin, ovomucoid andlysozyme. Some of the physicochemical properties of themajor egg-white proteins are presented in Table 1.

This review aims to present recent developments in egg-white protein functionality. Moreover, experimental evidenceof improved functional properties of egg-white proteins is cor-related to the findings of a recent patent application for a newindustrial spray drying process of egg-white [4].

2 Heat-induced structural changes ofglobular proteins

Denaturation of globular proteins under varying conditionscan lead to a great variety of properties, such as solubility, coa-

i 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Nahrung/Food 47 (2003) No. 6, pp. 369 – 376 369

Egg-white proteins are extensively utilised as food ingredients due totheir unique functional properties. Several attempts have been made inorder to improve the functional properties of egg-white proteins and toidentify the optimal formulations for unique food products. Experimen-tal data proves that controlled denaturation of egg-white proteins canhave a beneficial impact on various functional applications in the foodindustry such as emulsifying ability, heat stability, and gelation. This

review describes the effect of heat-induced denaturation on proteinstructure and functionality. Studies on the impact of Maillard reaction,which aim to elucidate the structure-function relationship of egg-whiteproteins, are presented. A novel approach which could be the basis forthe development of new methods aiming to improve the functional prop-erties of egg-white proteins is also discussed.

Correspondence: Dr. Lydia Campbell, School of Life Sciences, Her-iot-Watt University, Edinburgh EH14 4AS, ScotlandE-mail: [email protected]: +44-131-451-3009

Abbreviations: b-LG, b-lactoglobulin; OVA, ovalbumin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Keywords: Denaturation / Egg-white / Maillard reaction / Review /

Campbell et al.

370 Nahrung/Food 47 (2003) No. 6, pp. 369– 376 i 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

gulating (clotting), gelling, emulsifying and water-bindingproperties, each of which could result in a food product. Forinstance, despite the fact that both coagulation and gelationresult from aggregation between protein molecules there is adistinct textural difference between coagulated and gelledproducts. Coagulation can also be called “clotting” like itoccurs when denatured whey proteins randomly clumptogether when acidified and heat treated such as in Ricottacheese manufacture. Both gelation and coagulation is thoughtto be the result of a two-step process. The first step is a dena-turation process and the second an aggregation process. Theextent of unfolding following denaturation, the kinetics of theaggregation process and the nature of the interactions (cova-lent, non-covalent) appear to be important in determining thetype of gel or coagulant produced and its characteristics [5].

2.1 Denaturation

During the denaturation step, which can be induced by phy-sical (heating, high pressure) or chemical (enzymes) means,the protein molecule unfolds and adopts a different conforma-tional state compared to the native one. This results in theexposure of hydrophobic groups as well as “buried” sulfhydrylgroups. This “hydrophobic effect” at the secondary structurelevel usually means that there is a small decrease in the a-helixcomponent, and an increase in the proportion of b-sheet [6].These changes at the secondary structure of egg-white proteinscan have an impact on egg-white functionality, thus affectingproperties such as gelling ability and solubility. The denaturedprotein molecule possibly exhibits a more “reactive” confor-mational state compared to the untreated one, due to the factthat covalent and non-covalent interactions can now bemediated between neighbouring molecules, under suitable con-ditions. By using mild heat-treatments it has been shown thatglobular proteins in solution can undergo a conformationaltransition into a native like secondary structure without loss ofthe residual tertiary structure [7]. In more recent studies thisintermediate state or ‘molten globule’ state was also identifiedin heat-induced aggregation of ovalbumin molecules [8, 9]. Itis evident that the kinetics of the denaturation step are deter-mined by intrinsic factors (protein composition and concentra-tion) and extrinsic factors (heating temperature, heating time,pH, ionic strength, presence of other additives). For instance,proteins that are compact in shape and contain several disul-

fide bonds require fairly high thermal energy to disrupt thelinkages and to unfold the molecule. On the other hand, conal-bumin is the most thermolabile among the egg-white proteins,due to the absence of disulfide links in the molecule. That canbe a limitation to the thermal processing of egg-white, as con-albumin denatures and becomes insoluble at lower tempera-tures compared to the denaturation temperatures required fordenaturation of the other proteins present in the egg-white[10]. Therefore, to prevent coagulation, pasteurisation of egg-white in the standard commercial process takes place in thedry state. Conditions of the denaturation step are crucial interms of the structure the protein molecules adopt and this inturn determines their functional properties (structure–functionrelationship). The structural differences of egg-white proteinsheated in the dry state compared to proteins heated in solutionhave not been clearly elucidated.

2.2 Measurement of the denaturation degree

Various techniques are known in the art for measuring thedegree of denaturation of albumin and other proteins. In onesuch method the denaturation degree of albumin is determinedby measuring the quantity of reactive SH-groups [11]. Follow-ing heat induced denaturation the number of total SH-groups isexpected to decrease due to the SS-SH interchange and SH-SHoxidation. Turbidity measurements also give an indication ofprotein denaturation. Studies by Seideman et al. [12] haveshown that when liquid egg-white was heated at 588C for vary-ing times (at constant pH), the turbidity of the solution meas-ured as absorbance at 550 nm, increased with time of heating.It is speculated that this is an indirect method to measure dena-turation as turbidity actually measures the degree of aggrega-tion after denaturation has taken place. Ferreira et al. [13]developed an HPLC/UV method to monitor the extent of heat-induced denaturation of milk proteins during processing. Theextent of denaturation and loss of whey proteins when milkwas heated at specified temperatures was estimated by moni-toring the milk proteins remaining in a native form [13].Finally, UV circular dichroism, differential scanning calorime-try (DSC) and tryptophan autofluorescence spectroscopic stud-ies can provide evidence regarding the altered tertiary structureof proteins and thus indicate the extent of heat-induced dena-turation [14, 15].

Table 1. Composition and some physicochemical properties of the major egg-white proteins.

Protein Albumin(% dry mass basis)

pI Molecular mass(Da)

Td( 8C)

Characteristics

Ovalbumin 54.0 4.5 45 000 71.5 PhosphoglycoproteinOvotransferrin 12.0–13.6 6.1–6.6 76 000–80000 57.3 Binds metallic ionsOvomucoid 11.0 3.9–4.3 28 000 ND Inhibits trypsinOvomucin 3.5 4.5–5.0 110 000 ND Glycoprotein; viscousLysozyme 3.4–3.5 10.7 14 300–14600 81.5 Lyses some bacteriaOvoflavoprotein 0.8 4.0–4.1 32 000–35000 69–72 Binds riboflavinOvomacroglobulin 0.5 4.5–4.7 760 000–900000 69–72 GlycoproteinOvoinhibitor 1.5 5.1–5.2 44 000–49000 69–72 Inhibits trypsin and

chymotrypsinOvoglycoprotein 1.0 3.9 24 000–24400 69–72 GlycoproteinAvidin 0.5 9.5–10.0 55 000–68300 ND Binds biotin

Data were obtained from Gosset et al. [2] and Johnson et al. [3].pI, isoelectric point; Td, denaturation temperature; ND, not determined

Egg-white functional properties


3 Structural and functional propertiesof egg-white protein systems as aresult of heat treatment

3.1 Solubility

Solubility is related to the net free energy change from inter-action of hydrophobic and hydrophilic residues on the proteinsurface with the surrounding solvent [16]. Extreme heatingconditions of globulins in solution will eventually result in lossof tertiary structure, hence irreversible loss of solubility, whichcould lead to precipitation or coagulation. It is evident thatinsoluble proteins may not be desirable for food products andit is therefore important that heat denaturation is controlled soas not to result in a significant loss of protein solubility. How-ever, functional properties such as foaming or emulsifyingability are attributed to the protein surface activity and are notaffected by reduced solubility.

3.2 Gelation/coagulation

Following denaturation by heat treatment, the partiallyunfolded egg-white proteins are capable of interacting underfavourable conditions to form complexes. Studies of the gela-tion properties of egg-white, demonstrated that in a mixture ofall egg-white proteins, aggregation of the polypeptidesoccurred at two distinct coagulation temperature ranges. Thefirst range is from 61.58C–62.58C and the second range isfrom 71.08C–73.08C. The first coagulation temperature rangeinvolved conalbumin and possibly some other protein(s) whichwere denatured and partially aggregated, and coagulation wascompleted at the second temperature range [3].

The rate and order of aggregation of the egg-white proteinmolecules determines the final product of the process. Egg-white proteins are sometimes classified as coagulation typeproteins due to the relatively high percentage of hydrophobicamino acids composing the polypeptide chains of the mole-cules [2]. Coagulation, which in some cases is highly desirablein the food industry, is associated with decreased water holdingcapacity resulting from increased randomness of aggregates.Hermansson [17] reported that the addition of salt enhancesthe tendency of molecules toward random aggregation, andthereby the moisture is not bound to proteins.

The gelling ability of heated egg-white proteins is usuallythe result of balanced attractive and repulsive forces betweenthe protein molecules, resulting in a gel network in which

water is easily bound and retained. Gel formation is a compli-cated process and is generally affected by protein concentra-tion, quantity and state of water, ionic type and strength, heat-ing time/temperature, pH and interactions with other compo-nents [17]. Table 2 shows the impact of intrinsic (i. e., proteinconcentration) and extrinsic (i. e., ionic strength) factors on thetype of gel formed when egg-white proteins are heated at atemperature above the denaturation temperature (A61.58C).

Generally, gel formation is favoured under conditions thatreduce the electrostatic repulsive forces between the proteinmolecules. In other words, pH values neighbouring the isoelec-tric point (zero net charge) and high ionic strength (screeningthe electrostatic repulsive forces) can be promoting factors forthe formation of a 3-D network, provided that the protein con-centration is sufficiently high for the given conditions. Thediagram below describes the type and order of bond formationthat are involved at each step during the process of egg-whitegelation.

3.3 Foaming ability

The unique foaming abilities of egg-white are attributed tothe interaction between the various constituent proteins. Themain characteristics which define a good foaming agent can besummarised as follows: (i) to be able to adsorb rapidly at theair-water interface, (ii) to undergo rapid conformationalchange at the interface, and (iii) to form a cohesive viscoelasticfilm via intermolecular interactions [18]. The hierarchy ofegg-white proteins in terms of their importance for foaming isthe following: globulins, ovalbumin, ovotransferrin, lysozyme,ovomucoid, and ovomucin [18].

Graham and Philips [19] demonstrated that denatured oval-bumin possesses higher foaming power than native ovalbumin,because the proteins adsorb more easily to the air bubble sur-

Table 2. Factors affecting the texture of heat-induced ovalbumingels

Protein conc.(g/mL)

pH Ionic strength Effect on gelation

Very low (a0.05%) m pI Low No aggregationMedium (5%) m pI Low Linear polymersMedium (5%) m pI Medium Hard gelMedium (5%) pI High Soft gel

m means above or below isoelectric point. Data were obtained fromMine [18].

Figure 1. Schematic representationof heat-induced gelation of egg-whiteproteins. From Mine [18].

Campbell et al.


face. Furthermore, Relkin et al. [20] have shown that mildheat-treatments of ovalbumin can change the structure from a‘rigid’ conformational state to an intermediate stable partiallyunfolded state, also called the ‘molten globule’ state. Thesemodified protein structures exhibited enhanced foaming func-tionality such as enhanced rate surface tension decrease andincrease in foam stability (reduced liquid drainage). That isprobably due to increased flexibility and surface hydrophobi-city exhibited by ovalbumin in the “molten globule” state. Ifthe solutions of partially unfolded ovalbumin are heated atmore extreme conditions, they are unstable and coagulationensues, thereby reducing the foaming power.

3.4 Effect of spray drying

Using conventional spray-drying procedures, native egg-white proteins are not subjected to denaturation [21]. Hence itcan be assumed that spray drying of the denatured albumenwould not lead to further denaturation or coagulation. In thestandard process for spray drying of egg-white, the product isnot pasteurised prior to spray drying, because the egg proteinswill coagulate at temperatures higher than 578C. It is wellknown that if untreated egg-white is spray dried, the producthas poor whipping properties and develops a brown colour andunpleasant taste, even after short periods of storage at ambienttemperature [22]. It has been shown that the mechanismresponsible is the reaction between proteins and covalentlybound sugar, which results in a brown colour (Maillard reac-tion) [22]. The egg-white in the conventional spray drying pro-cess is therefore submitted to a de-sugaring process step priorto spray drying. The process that removes these sugars takes5–6 h at 308C and it involves fermentation by bacteria oryeast. After spray drying, the egg-white powder is pasteurisedat approximately 548C for 7 to 10 days in a sealed container.Thus, the current commercial process may take as long as twoto three weeks to produce shelf stable egg-white powder.

4 Functional properties of egg-whiteheated in the dry state

4.1 Solubility

Kato et al. [23] investigated the effect of heating egg-whitein the dry state on its functional properties. Egg-white wastreated at 808C for a period of up to 7 days (7.5% humidity).Surface hydrophobicity increased with increases of heatingtime in the dry state and protein aggregates were formedthrough hydrophobic interactions and disulfide bonds. As aresult, we would expect a decrease in the solubility of egg-white proteins, due to the fact that more “water hating” groupsare exposed on the protein surface following denaturation andbecause of the aggregate formation. Interestingly, the resultsindicated that there was no effect for heating in the dry state onthe solubility of egg-white proteins. It was suggested that a bal-ance between hydrophilic and lipophilic surface groups musthave occurred during the heating process, which did not haveany significant effect on solubility of the resulting proteins.

4.2 Heat-induced gelling ability

Several studies indicate a correlation between total and/orsurface SH groups of egg-white heated in the dry state and thegelling properties (after dissolving the powder in water andheat treatment). Ovalbumin (OVA) contains four sulfhydryl

(SH) groups and one disulfide (SS) group per molecule in thenative state. Matsudomi et al. [24] investigated the effect ofheating on dried ovalbumin (808C, 7 days) with respect to thetotal and surface number of SH groups. Their results indicate adecrease in the total number of SH groups and an increase inthe number of surface SH groups, as temperature increased.Most probably, the SH groups are exposed to the exterior ofthe protein molecule, as a result of heat denaturation and areallowed to interact inter- or intramolecularly. The formation ofthe disulfide links through SH-SS exchange reactions and SH-SH oxidation is suspected to play a role to the excellent gellingproperties of egg-white heated in the dry state.

As seen from Fig. 2, the total SH-groups are reduced byapproximately 0.5 mol per OVA molecule after 10 days ofheating. This means that the formation of disulfide bridgesoccurs to a limited extent. On the other hand, the number ofsurface SH groups slightly increases after 10 days of heating.This can be explained by the fact according to which nearly allof the methionine residues (SH-groups) that are exposed to theexterior of the molecule are involved in disulfide bond forma-tion.

In order to assess the type of interactions which are respon-sible for the firm, uniform gel generated by egg-white heatedin the dry state, Matsudomi et al. carried out sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) ana-lysis on the protein samples (Fig. 3). As revealed, the bandscorresponding to aggregates were more intense with increasedtime of heating. Furthermore, some of the aggregated proteinpopulations were not affected by the presence of 2-mercap-toethanol (ME), suggesting that tight interactions, other thandisulfide bonding contribute to the formation of the firm gel.

4.3 Emulsifying ability

The impact of heating in the dry state (808C, 10 days, 7.5%humidity) on emulsifying ability of egg-white proteins wasinvestigated [23]. There was a linear correlation betweenincreased emulsifying activity and longer heating time in thedry state. Furthermore, the emulsion stability also increased byheating in the dry state. Kato et al. [23] stated that increasedsurface hydrophobicity resulting from heating in the dry stateaccounted for the improvement of functional properties suchas emulsifying capacity and emulsion stability. That is due toincreased flexibility and surface hydrophobicity of egg-whiteprotein molecules following heat-denaturation which enable

Figure 2. Changes of total and surface sulfhydryl (SH) groups indry-heated ovalbumin (OVA) at 80 8C for various periods of time.(0) Total SH, (9) Surface SH. Data are the average of triplicate meas-urements. From Matsudomi et al. [24].



them to adsorb better at the oil-in water interface with the“exposed” hydrophobic residues in contact with the oil phase.

Kato et al. [23] demonstrated that heating egg-white in thedry state improves the foaming properties of the proteins. Bothfoaming power and stability were affected by heating in thedry state and it was shown that the above properties wereenhanced with increases of heating time in the dry state. Theresults include samples that had been subjected to heat for aprolonged period of time (10 days) and indicate that egg-whiteproteins can improve their foaming abilities even after exten-sive heat treatment [23].

5 Combined effects of sugar and salton structural and functionalproperties of heat-treated egg-whiteproteins: a novel approach

To date there does not appear to have been fundamentalscientific studies on the combined effects of sugar and salt onegg-white denaturation and aggregation upon heating. Thepatent by Campbell and Tr�ck [26] describes the heat treat-ment of whey protein in the presence of sugar and salt, whichrendered the denatured protein stable against acidification.This simplified the process for a mayonnaise-type manufacturewith whey protein. When the same principle was applied toegg-white the functionality was improved in such a way that itcould serve as a fat replacement material in mayonnaise-typeemulsions [4]. Suitable conditions for the heat treatment weredetermined by means of monitoring the degree of aggregationof the egg-white proteins by monitoring increase in turbidity ofthe solution. The aggregation process was measured quantita-tively by determination of increase in turbidity of the egg-white solution upon heating, by means of a modified methodof Seideman et al. [12]. Thermal treatment conditions wereselected so as to obtain between 60 to 80% denaturation ascompared to the unheated protein. The partially unfolded egg-white protein mixture has improved emulsifying and waterbinding properties. Egg-yolk can be partially replaced in emul-sions like mayonnaise and dressings, thereby lowering choles-terol content and reducing costs [4].

5.1 Heat stability

Depending on the reducing sugar (i. e., glucose) and saltconcentration, the pasteurisation temperatures of egg-whiteprior to spray drying could be as high as 648C, thereby elimi-nating the need for pasteurisation of the powder after spray

drying. The combination of reducing sugar and salt had asynergistic effect on the delay of coagulation whilst keepingthe denatured protein in solution [4]. The use of salt for stabili-sation of the proteins is optional whereas the use of a carbonylcompound (i. e., reducing sugar) is essential to affect denatura-tion without immediate coagulation [4].

5.2 Egg-white properties after spray-drying

It was found that by carefully controlling the degree ofunfolding of egg-white proteins (by measurement of the aggre-gation process) when heated in the presence of glucose (andsalt), the browning process is significantly reduced after spraydrying and the product can be stored for up to a year at roomtemperature, without changing colour [4]. Similar inhibition ofthe Maillard reaction by spray drying of egg-white in the pres-ence of sugar is described by Kline et al. [22]. This patent,however, does not include a pasteurisation step in the process,neither before, nor after the spray-drying step, thereby lackingin microbial safety control of the product.

The denatured, sugared, egg-white powder does not turnbrown for up to one years’ storage at ambient temperature [4].This is surprising since one would expect the Maillard reactionto be even more severe, because the covalently bound sugarhas not been removed and it was heated in the presence of glu-cose, which is a reducing sugar. (The Maillard reaction occursmore commonly with reducing sugars than with non-reducingsugars ). It may well be asked how can such sugars be effectiveto stabilise the product when conventional treatments involvethe deliberate elimination of reducing sugars by fermentation.One explanation given by Kline et al. [22] is that the smallamount of water normally present in the dried product, whichis required for the Maillard reaction to occur, is bound by the aadditional carbohydrates so that the reaction is hindered due toa lack of moisture.

6 The Maillard reaction: basis forimproved functional properties ofegg-white treated as stated in thenovel approach

Egg-white proteins treated as described in Section 5 appearto have improved functional properties such as emulsifyingability and water binding capacity. However, up to date there isno direct evidence to link the structural changes occurring bythe Maillard reaction and the improved functional properties ofthe proteins after treatment. Several investigators stated that

Figure 3. Electrophoretic patterns of dry-heated OVA at 80 8C for various periods oftime. (A) Native PAGE, (B) SDS-PAGEwithout 2-mercaptoethanol (ME), (C) SDS-PAGE with ME. The lane numbers showheating time (days). Lane M indicates mar-ker roteins (phosphorylase, 97 kDa; bovineserum albumin, 66 kDa; aldolase, 42 kDa;carbonic anhydrase, 30 kDa; soyabean tryp-sin inhibitor, 20 kDa). Adapted from Matsu-domi et al. [24].

Campbell et al.


the functional properties of egg-white proteins can beimproved by conjugation with a polysaccharide moiety [28–33]. In most of the cases the treatment of proteins is differentto the process described in Section 5. Nevertheless, themechanism responsible for improved functional properties ofegg-white might be similar. The findings of the investigationsregarding the protein-polysaccharide conjugates are discussedin the following section.

6.1 Effect of heating in the presence of sugars onglobular protein structure

It is well documented that sucrose stabilises globular pro-teins such as egg-white and whey protein against thermal dena-turation [34, 35]. As a result, the denaturation temperature ofglobular proteins increases in the presence of sugars. Heatingof protein solutions in the presence of sugars can result in theformation of covalent protein-sugar complexes via the amino-carbonyl side chains (Maillard reaction) [35]. This increasesthe hydrophilic character of the proteins and changes func-tional properties (i. e., increased solubility). Moreover, thehydrodynamic properties of the molecule are altered as theshape from compact shifts to a more branched conformation.As a result, the solution properties (i. e., viscosity) are affected.

The Maillard reaction has been extensively studied in var-ious protein systems, among which are whey proteins. Wheyproteins are undoubtedly among the best-characterised proteinsin food. The major whey proteins are b-lactoglobulin, a-lacto-globulin and bovine serum albumin. The mechanism of heat-induced denaturation and aggregation of whey proteins in thepresence of a reducing sugar shows some exceptional charac-teristics and, therefore, cannot be considered as a model pro-tein system. However, whey proteins are industrially relevantto egg-white proteins due the fact that they both consist of amixture of globulins and they are used for common purposesin food (i. e., gelling agents). The molecular basis of whey pro-tein functionality has been extensively studied [36]. There nowexists detailed knowledge of the molecular structure of heated

b-lactoglobulin, such as chemical modification (glycation orlactosylation), conformational rearrangement, aggregation andnetwork formation [36]. Morgan et al. [37] studied the glyca-tion of b-lactoglobulin when heated in solution with lactoseand also heated as a dry powder in the presence of lactose. Theresults showed that the dry-state glycation did not significantlyalter the conformation of the protein, whereas the treatment insolution led to significant structural changes. Analyses of poly-peptide chain modifications by electrospray ionization-massspectrometry (ESI-MS) showed that covalent complexesbetween lactose and the polypeptides were formed in bothtypes of heat treatment. However, only the protein and lactoseheated in solution showed the formation of high-molecular-weight aggregates or polymers when analysed by size-exclu-sion chromatography (Fig. 5). SDS-PAGE analyses showedthat the complexes consist of disulfide-linked homodimers.Similar thiol/disulfide interchange reactions were not observedwhen the protein was heated in the dry state and it was con-cluded that covalent aggregation of b-lactoglobulin (b-LG)was blocked at the dimer step. Non-covalent polymerisation ofthe unfolded homodimers was demonstrated [38].

6.2 Impact of Maillard reaction on the functionalproperties of egg-white proteins

Covalent attachment of sugar residues, using mild, non-denaturing heat-treatment, has been reported to increase theheat stability of globular proteins [27] and to improve emulsi-fying ability [28]. Although sugars protect proteins from ther-mal denaturation by enhancing protein-protein interactions,once denatured they appear to have improved functional prop-erties due to the enhanced amphiphilic character caused by thesugar attachment.

6.3 Solubility

Egg-white proteins treated as described in the patent byCampbell and Truck were still soluble after spray drying [4].This phenomenon is supported by other experimental data,which revealed that the polymerisation of OVA resulting fromglucose and glucose-6-phosphate attachment to the proteinbackbone (508C, 0–5 days, 65% relative humidity) gave riseto a sugar modified protein molecule with improved heatstability [27]. At high solution concentration (5%) the proteinwas still completely soluble and transparent even after treat-ment at extreme conditions (1008C, 10 min). On the otherhand, the ovalbumin molecules, which had not been modifiedby sugar attachment were subjected to the same heat treatmentand exhibited a dramatic loss in solubility. Other investigators

Figure 4. Schematic representation of the Maillard reaction.

Figure 5. Size-exclusionchromatography profiles of(A) native and dry-way treatedb-LG; (B) b-LG heated in solu-tion without lactose for 130 h(control sample); (C) b-LGheated in solution with lactosefor 130 h. HMW, high-molecu-lar-weight species. From Mor-gan et al. [37].



have also reported that egg-white proteins were converted intostable and soluble forms by binding with polysaccharides [29–31]. We may therefore suggest that the Maillard reaction canhave a positive impact on the solubility of heat-denatured glob-ular proteins. This may be due to the fact that protein mole-cules become less hydrophobic after covalent modificationwith sugar molecules. This means that more polar groups arepresent on the surface area of the protein backbone, resultingin enhanced solubility in aqueous environment.

6.4 Gelation

Numerous experimental evidence indicates that glycosyla-tion of proteins with reducing sugars results in improved func-tional properties of food proteins. Gelation is among theseproperties. Handa and Kuroda [32] investigated the mode ofinteraction between dried egg-white and glucose through theMaillard reaction, which results in improved gelling propertyof dried egg-white under certain controlled conditions [32].De-sugared dried egg-white was incubated with glucose at var-ious heating times (558C, 0–12 days, 35% relative humidity)[32]. Native PAGE profiles indicated an increase in the netnegative charge of the proteins suggesting an attachment of thereducing sugar to a lysine residue (positively charged) of theegg-white protein. Furthermore, SDS-PAGE analysis revealedan increase in the dried egg-white molecular weight resultingfrom the glucose attachment. The degree of polymerisationwas found to increase with increasing heating time. Bothmonomeric and polymeric glycosylated protein molecule popu-lations appeared. The latter were visualised as aggregates on theSDS-PAGE, a proportion of which could not be disrupted underdenaturing conditions. This suggests the formation of nondisul-fide covalent bonding. Conclusively, we could point out thatprotein-sugar adducts resulting from the Maillard reaction, areavailable for further reaction either with unreacted proteinmonomer or with other protein adducts [39]. Disulfide bondingand nondisulfide covalent interactions are possibly involved inthe polymerisation of egg-white proteins, and the resultingstructures may contribute to the excellent gelling properties ofsugared dried egg-white. For comparison purposes, Handa andKuroda examined the gelling properties of desugared egg-whiteproteins subjected to the same heat treatment (558C). It wasconcluded that the Maillard reaction significantly improves thegelling ability of egg-white proteins.

6.5 Emulsifying properties

Aoki et al. [30] reported that the emulsifying activity ofOVA was improved by conjugation with glucuronic acid(508C, up to 1 day, 65% relative humidity) through the Mail-lard reaction. The effect on the structure of OVA was similar tothe one generated by succinylation. Succinylation is a chemicalmodification, which converts cationic amino groups to anionicresidues, thus improving their functional properties. Since suc-cinylation is a chemical modification it is highly desirable toachieve a similar modification to food proteins through amechanism that is naturally occurring such as Maillard reac-tion. Aoki et al. [30] stated that the emulsifying activity of glu-curonic acid-OVA conjugate (GlcUA-OVA) was 3.2 times thatof untreated ovalbumin, as shown in Fig. 6. The emulsifyingactivity was determined by the method of Pearce and Kinsella[40] that measures the turbidity of a water in oil emulsiondiluted in 10% SDS.

Moreover, Kato et al. [29] studied the effect of the Maillardreaction on the emulsifying ability of egg-white by attaching

the mannose hydrolysate of guar gum (galactomannan) [29].The conjugate formation was carried out by heating in the drystate. The experimental results indicated much better emulsify-ing activity and emulsion stability compared to untreated(without galactomannan) samples. Even more, the galactoman-nan conjugated egg-white proteins exhibited a better emulsify-ing activity compared to commercial emulsifiers. Again, theemulsifying activity was retained in a salt environment (0.2 M

NaCl) and under extreme conditions (acidic pH, high tempera-ture), which may render the product suitable for industrial pro-cessing. It was concluded that the attachment of hydrophilicmoieties (polysaccharides) to the protein chain is crucial forthe improved emulsifying properties of the conjugates. Theamphiphilic conjugates adsorb better at the oil-in-water inter-face with the hydrophobic side chains oriented to the oil phaseand the hydrophilic side chains oriented to the water phase.

A similar mechanism might be responsible for the improvedemulsifying properties of denatured, sugared, spray dried egg-white described by Campbell [4]. The Maillard conjugatescould “mimic” the amphiphilic character of egg-yolk lipopro-teins, and thus rendering efficient the partial substitution of thelatter by non-cholesterol containing components (sugared,spray dried egg-white proteins).

6.6 Foaming ability

The effect of glycosylation on foaming properties of egg-white has attracted less attention. Chevalier et al. [41] investi-gated the effect of Maillard reaction on the functional proper-ties of b-LG glycated (608C, 3 days) with several sugars (ara-binose, galactose, glucose, lactose, rhamnose, ribose) [41].The results indicated that glycosylation with moderately reac-tive sugars such as glucose and galactose improved the foam-ing ability of b-LG to a higher extent, compared with the othersugars. That means that the type of sugar used for protein-polysaccharide conjugation may be important with respect tothe final functional properties of the conjugate [41]. It is evi-dent that by carefully controlling the precise concentrationsand heating profiles of protein sugar adducts, we may be ableto manipulate the property of foaming in a highly desirableway [4]. Up to date, functional properties of Maillard reactedegg-white proteins such as foaming capacity and stability meritinvestigation.

Figure 6. Emulsifying activity of untreated OVA and GlcUA-OVA-conjugates. OVA was incubated at 50 8C with GlcUA for a period of upto two days and emulsifying activity index was determined. Each datashows mean value of the two determinations, of which deviation wasless than 0.050 absorbance units. From Aoki et al. [30].

Campbell et al.


7 Conclusions

Egg-white proteins have been extensively utilised as foodingredients thanks to their functional properties. The function-ality of egg-white proteins in the food industry is associatedwith the molecular basis of the structural changes induced bythe physicochemical procedures employed during processing.Each procedure has a different impact on the molecular struc-ture of the egg-white proteins, thus affecting the functionalproperties in a distinct way. The mechanisms involved in thestructure-function relationship of the globular proteins are notyet fully understood.

This article provides a summary of the existing practical evi-dence that egg-white proteins when denatured have improvedfunctional properties as related to food. The concept accordingto which egg-white proteins are capable of existing in a “mol-ten globule state” adds a new perception on the effect of pro-cessing conditions on egg-white functionality. The patent byCampbell [4] describes a novel approach to alter the physico-chemical properties of egg-white proteins in solution. To datethere does not appear to have been a study of the combinedeffects of sugar, salt and pH on egg protein denaturation andaggregation upon heating. The presence of externally addedcarbohydrate appears to be essential to keep the denatured pro-tein in an intermediate state of suspension, under controlledexperimental conditions. The role of salt (NaCl) in the delay ofalbumen coagulation when heated in the presence of sugar isnot fully understood.

The exact molecular structure and dynamics of the protein-carbohydrate complex needs to be characterised. Althoughdenaturation of the major egg-white proteins (apart from theheat stable proteins ovomucin and ovomucoid) occurs, thecomplex remains in suspension while there is a delay in proteincoagulation. The protein-carbohydrate complex can be pas-teurised at higher temperatures to eliminate any microbiologi-cal threat and its spray drying process can be simplified, whichcould result in substantial cost savings in egg-white process-ing. Initial analysis indicates that the end product has improvedemulsifying, water binding and (probably) foaming properties,which have potential for cost saving of egg raw materials infood production. Further experimental and scientific studiesare required to substantiate and elucidate the practical evi-dence for its improved functional properties in food. Moreover,further research is required to clarify the structural changes ona molecular basis that occur in such a multiprotein systemsuch as egg-white under the controlled manipulation of numer-ous variables (salt, sugar, pH, temperature).

8 References[1] McWilliams, M., Foods Experimental Perspectives, Prentice-

Hall, New York 1997.[2] Gosset, P. W., Rizvi, S. S. H., Baker, R. C., Food Technol. 1984,

38, 67–96.[3] Johnson, T. M., Zabik, M. E., Poultry Sci. 1981, 60, 2071–2083.[4] Campbell, L., Patent PCT WO 02/49442 A1 (2002).[5] Mulvihill, D. M., Rector, D., Kinsella, J. E., Food Hydrocolloids

1990, 4, 267–276.[6] Gosal, W. S., Murphy, B. R., Curr. Opin. Colloid Interface Sci.

2000, 5, 188–194.

[7] Kuwajima, K., Hiraoka, Y., Ikeguchi, M., Sugai, S., Biochemistry1985, 24, 874.

[8] Hirose, H., Trends Food Sci. Technol. 1993, 4, 48.[9] Hagolle, N., Relkin, P., Dalgleish, D. G., Food Hydrocolloids

1997, 11, 311.[10] Perez, O. E., Pilosof, A. M. R., in: Dickinson, E., Vliet, T. V.

(Eds.), Food Colloids, Biopolymers and Biomaterials, RSC2003, 119–132.

[11] Kitabake, N., Doi, E., J. Agric. Food Chem. 1987, 35, 953–975.[12] Seideman, W. E., Cotterill, O. J., Funk, E. M., Poultry Sci. 1963,

42, 406.[13] Ferreira, I., Mendes, E., Ferreira, M. A., Anal. Sci. 2001, 17,

449–501.[14] Marangoni, A. G., Barbut, S., McGauley, S. E., Marcone, M.,

Narine, S. S., Food Hydrocolloids 2000, 14, 61–74.[15] Deng, Y., Rosenvold, K., Karlsson, A. H., Horn, P., Hedegaard, J.,

Stenffensen, L., Andersen, H. J., J. Food Sci. 2002, 67, 1642–1647.

[16] Mine, Y., Food Res. Int. 1996, 29, 155–161.[17] Hermansson, A. M., J. Food Sci. 1982a, 47, 1960–1964.[18] Mine, Y., Trends Food Sci. Technol. 1995, 6, 225–232.[19] Graham, D. E., Philip, M., in: Akers, R. J. (Ed.), Forms, Aca-

demic Press, London 1976, 237–255.[20] Relkin, P., Hagolle, N., Dalgleish, D. G., Laurey, B., Colloid and

Surfaces B: Biointerfaces 1999, 12, 409–416.[21] Masters, K., Spray Drying Handbook, Longman Scientific &

Technical, Harlow, UK 1991, pp. 51–52.[22] Kline, L., Cerrito, E., Sugihara, T. F., Meehan, R. J., Patent US

3170804 (1965).[23] Kato, A., Ibrahim, A. R., Watanabe, H., Honma, K., Kobayashi,

K., J. Agric. Food Chem. 1989, 37, 433–437.[24] Matsudomi, N., Takahashi, H., Miyata, T., Food Res. Int. 2001,

34, 229–235.[25] Camejo, G., Golacio, G., Rapport, R. M., J. Lipid Res. 1968, 9,

562.[26] Campbell, L., Truck H. U., European Patent EP0788747A1

(1997).[27] Kato, Y., Aoki, T., Kato, N., Nakamura, R., Matsuda, T., J. Agric.

Food Chem. 1995, 43, 301–305.[28] Miyaguchi, Y., Tsutsumi, M., Nagayama, K., J. Jap. Soc. Food

Sci. Technol. 1999, 46, 514–520.[29] Kato, A., Minaki, K., Kobayashi, K., J. Agric. Food Chem. 1993,

41, 540–543.[30] Aoki, T., Hiidone, Y., Kitahata, K., Sugimoto, Y., Ibrahim, H.,

R., Kato, Y., Food Res. Int. 1999, 32, 129–133.[31] Delben, F., Stefanich, S., Food Hydrocolloids 1998, 12, 291–

299.[32] Handa, A., Kuroda, N., J. Agric. Food Chem. 1999, 47, 1845–

1850.[33] Lee, J. C., Timasheff, S. N., J. Biol. Chem. 1981, 256, 7193–

7201.[34] Jou, K. D., Harper, W. J., Milchwissenschaft 1996, 51, 509–512.[35] Friedman, M., J. Agric. Food Chem. 1996, 44, 631–653.[36] Holt, C., Int. J. Food Sci. Technol. 1999, 34, 407–408.[37] Morgan, F., Moll�, D., Gw�n�le, H., Venien, A., Leonil, J., Peltre,

G., Levieux, D., Maubois, J. L., Bouhallab, S., Int. J. Food Sci.Technol. 1999, 34, 429–435.

[38] Morgan, F., L�onil, J., Moll�, D., Bouhallab, S., J. Agric. FoodChem. 1999, 47, 83–91.

[39] Fayle, S. E., Healy, J. P., Brown, P. A., Reid, E. A., Gerrard, J. A.,Ames, J. M., Electrophoresis 2001, 22, 1518–1525.

[40] Pearce, K. N., Kinsella, J. E., J. Agric. Food Chem. 1978, 26,716–723.

[41] Chevalier, F., Chobert, J. M., Popineau, Y., Georgette, M., Haert-le, N., Haertle, T., Int. Dairy J. 2001, 11, 145–152.

Received September 19, 2002Accepted June 20, 2003

modification of functional properties of egg-white proteins

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