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FOODHYDROCOLLOIDS

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Food Hydrocolloids 21 (2007) 607–616

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Whey protein–maltodextrin conjugates as emulsifying agents:An alternative to gum arabic

Mahmood Akhtar, Eric Dickinson�

Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK

Received 4 April 2005; accepted 29 July 2005

Abstract

The emulsifying properties of covalent complexes of maltodextrin (MD) with whey protein (WP) isolate have been investigated under

both acidic and high electrolyte concentration conditions in systems containing medium-chain triglyceride oil or orange oil. Covalent

coupling of protein to polysaccharide was achieved by dry-heat treatment of a protein+polysaccharide mixture for up to 2 h. It was

confirmed by SDS-polyacrylamide gel electrophoresis that the WP does become directly linked to the MD. Analysis of droplet-size

distributions has shown that this covalent linking of MD to WP leads to a very substantial enhancement in the protein emulsifying

behaviour under both acidic and neutral conditions. Analogous dry-heating treatment of MD with soy protein does not have this

positive effect. A whey protein–MD conjugate WP–MD19, made fromMD (DE ¼ 19) of intermediate mean molecular weight (8.7 kDa),

has been found to be capable of producing fine emulsion droplets (0.5–1 mm) with either triglyceride oil or orange oil. Optimized

WP–MD19 conjugates can produce fine stable emulsions (20 vol% oil) at 2wt% emulsifier content, whereas the equivalent emulsion

made with gum arabic requires a 20–30wt% level of emulsifier. A WP–MD19 conjugate of protein/polysaccharide ratio 1:2 or 1:3 is

effective in stabilizing low-pH emulsions of a commercial flavour oil (containing a weighting agent) over a storage period of several

weeks, with no visible precipitation or phase separation when mixed with colouring agents, either before or after extensive emulsion

dilution.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Whey protein; Protein–polysaccharide complex; Emulsion stability; Maltodextrin; Orange oil; Gum arabic; Colouring agent

1. Introduction

Proteins are efficient emulsifying agents and stabilizers offood oil-in-water emulsions under conditions where solu-bility is good and the aqueous phase environment issuitable for effective steric and electrostatic stabilization(Dickinson & Stainsby, 1982). On the other hand,emulsifying behaviour can become poor under certainconditions due to aggregation or precipitation of proteinand the associated loss of colloidal stabilizing character-istics. This instability is typically most pronounced at pHvalues close to the protein’s isoelectric point and at highconcentrations of electrolytes (Damodaran, 1996). It canalso occur in the presence of other species like colouringagents which bind strongly to adsorbed proteins, thereby

ee front matter r 2006 Elsevier Ltd. All rights reserved.

odhyd.2005.07.014

ing author. Tel.: +44 113 343 2956; fax: +44 113 343 2982.

ess: e.dickinson@food.leeds.ac.uk (E. Dickinson).

changing the net molecular charge and the balance ofinterfacial hydrophilic and hydrophobic interactions.It is now well-recognized that impressive improvements

in protein solubility and interfacial functionality can beachieved via the complexation and covalent linking ofproteins to polysaccharides (Dickinson, 1995; Ledward,1994; Samant, Singhal, Kulkarni, & Rege, 1993; Schmitt,Sanchez, Desobry-Banon, & Hardy, 1998; Syrbe, Bauer, &Klostermeyer, 1998). In particular, for the stabilization ofemulsions and foams, it has been demonstrated thatMaillard-type conjugates produced by the dry-heating ofa mixture of these two kinds of biopolymers canhave excellent functional properties (Akhtar & Dickinson,2003; Chevalier, Chobert, Popineau, Nicolas, & Haertle,2001; Dickinson & Galazka, 1991, 1992; Dickinson & Izgi,1996; Dickinson & Semenova, 1992; Diftis & Kiosseoglou,2003; Dunlap & Cote, 2005; Einhorn-Stoll, Ulbrich, Sever,& Kunzek, 2005; Kato, 1996; Kato, Sasaki, Furuta, &

ARTICLE IN PRESSM. Akhtar, E. Dickinson / Food Hydrocolloids 21 (2007) 607–616608

Kobayashi, 1990, 1992; Mishra, Mann, & Joshi, 2001;Morris, Sims, Robertson, & Furneaux, 2004; Nagasawa,Takahashi, & Hattori, 1996; Neirynck, van der Meeren,Bayarri Gorbe, Dierckx, & Dewettinck, 2004; Shepherd,Robertson, & Ofman, 2000). The formation of these high-molecular-weight glycoprotein conjugates combines thecharacteristic property of proteins to adsorb strongly to theoil–water (or air–water) interface with the characteristicproperty of the polysaccharide for solvation by theaqueous phase medium (Dickinson, 1995; Dickinson &Galazka, 1991). Such studies have shown that theeffectiveness of the conjugate as an emulsifier/stabilizer inmodel systems is dependant inter alia on the protein/polysaccharide ratio and the polysaccharide molecularweight. This kind of conjugate, which is ‘natural’, non-toxic and relatively simple to prepare, has for some timebeen recognized (Dickinson, 1993) as having significantpotential for exploitation in food-related emulsificationapplications.

Most of the previous investigations of emulsion stabili-zation by protein–polysaccharide conjugates have beenconcerned with model systems based on hydrocarbon oilsor triglyceride oils under nearly ideal aqueous solutionconditions. The present paper aims to demonstrate thepotential of this type of conjugate for making andstabilizing more challenging and complex emulsion systemsof low pH and raised ionic strength. The compositionalconditions are focused here towards carbonated beveragesystems based on an emulsified flavour oil in the presenceof a commercial colouring agent. To improve the potentialfor commercial viability, the conjugate emulsifiers aremade here from two relatively inexpensive biopolymeringredients, whey protein (WP) and maltodextrin (MD).Through our choice of ingredients and treatment condi-tions, we avoid here the very extended dry-heating times(several days) of some earlier studies. We furthermoreinvestigate the feasibility of replacing the WP by soyaprotein. Gum arabic (GA) is chosen as the referenceemulsifier because of its common use for encapsulation offlavour oils and for their emulsification in beverageemulsions (Islam, Phillips, Sljivo, Snowden, & Williams,1997; McNamee, O’Riordan, & O’Sullivan, 1998). Due tounpredictable fluctuations in supply and price of GA(Seisun, 2002), its replacement by alternative emulsifyingingredients remains a continuing source of interest to thefood industry.

The influence of the molecular weight of the MD on theemulsifying properties of whey protein–maltodextrin(WP–MD) conjugates has been investigated under bothneutral and acidic conditions. We compare three differentoil phases: a triglyceride oil, an orange oil, and acommercial flavour oil formulation containing orange oiland a weighting agent. The effect of the mass ratio ofprotein to polysaccharide on the emulsion stability hasbeen investigated. The better performing WP–MD con-jugates were tested in a model beverage formulation. Forconsistency throughout, all emulsions prepared with the

WP–MD conjugates initially contain the same amount ofoil (20 vol%) and a well-defined content of emulsifier (say,2wt%) (i.e. for the 1:3 ratio conjugate, comprising 0.5wt%protein+1.5 wt% MD).

2. Materials and methods

2.1. Materials

The commercial WP isolate sample, BiPro, obtainedfrom Davisco Foods International (Le Sueur, MN, USA),had been manufactured from fresh sweet whey, andconcentrated and spray dried into a homogenous, semi-hygroscopic, lactose-free powder. Three MD samples ofdifferent dextrose equivalent (DE), and hence differentvalues of the average molecular weight Mw, were pur-chased from Roquette (UK) Ltd: DE ¼ 2 (Mw ¼ 280 kDa),DE ¼ 19 (Mw ¼ 8.7 kDa), and DE ¼ 47 (Mw ¼ 2 kDa).The medium-chain triglyceride oil (MCT oil), orange oiland ‘oil mixture’ were provided by Quest International(UK). The ‘oil mixture’ was a commercial formulationcontaining (mainly) orange oil mixed with a ‘weightingagent’ to match closely the oil density to that of water. GA(Instantgum ASIRX 40.830) was obtained from ColloıdsNaturels International France. Sodium caseinate (5.2 wt%moisture, 0.05wt% calcium) was purchased from deMelkindustrie (Veghel, The Netherlands). A sample ofsoy protein (SP) isolate was supplied by Quest Interna-tional (UK). Sodium lactate (SL) solution (about 50%, LotK24888657) was purchased from BDH Laboratory Sup-plies (UK). Sodium benzoate (99%) and sodium citrate(99.5%) were purchased from Aldrich Chemicals (UK).The colouring agents, Ponceau 4R and Sunset yellow FCF,were obtained from Flevo Chemie (The Netherlands).

2.2. Preparation of conjugates

The WP (or SP) and MD were brought into goodcontact by dissolving them in distilled water at the specifiedweight ratio (e.g. 1:2 or 1:3). The samples were freeze-driedto remove water, and were ground to make a whitepowder. The freeze-drying process by itself did not lead toany detectable protein–polysaccharide association.Covalent coupling between protein lysine residues and

the reducing groups on the carbohydrate was achieved byheating the freeze-dried powder. Each sample was placed ina pre-heated desiccator at relative humidity of 79%.Following a typical incubation period of, say, 2 h at80 1C, the powder exhibited the light brownish appearancethat is typical of a moderate degree of non-enzymaticbrowning. For the same degree of browning, the incuba-tion temperature could be reduced by increasing theincubation time. To determine the effect of pH onconjugate solubility/dispersibility, the optical densities ofsolutions at various concentrations were measured atroom temperature using a Klett–Summerson Photoelectric

ARTICLE IN PRESSM. Akhtar, E. Dickinson / Food Hydrocolloids 21 (2007) 607–616 609

Colorimeter (model 800-3) with operating wavelengthrange of 640–700 nm.

Table 1 gives the abbreviations used in this paper toindicate the protein samples and the different protein–polysaccharide conjugates investigated.

2.3. SDS-polyacrylamide gel electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophor-esis (SDS-PAGE) was performed according to the methodof Laemmli (1970) using a 12% acrylamide separating geland a 6% stacking gel containing 0.1% SDS. Samples(30 ml, 0.1% of protein) were prepared in a 0.01MTris–glycine buffer (pH ¼ 8.8) containing 1% SDS. Elec-trophoresis was carried out at a constant current of 15mAon a gel for 2 h in a Tris–glycine buffer containing 0.1%SDS. The gel sheets were stained for both protein (0.2%Coomassie brilliant blue G-250) and carbohydrate (0.5%periodate–fuchin solution) (Zacharius, Zell, Morrison, &Woodlock, 1969).

2.4. Emulsion preparation and stability

The aqueous buffer was prepared using double-distilledwater, citric acid (50%), benzoic acid (25%), andpotassium metabisulfite (15%). Protein or protein/poly-saccharide conjugate was added slowly to the buffersolution at ca. 22 1C with gentle stirring. The pH of theresulting protein solution was adjusted to pH 3.2 by addinga few drops of 1M NaOH. The subsequently reported pHvalues refer to the pH of the protein solution beforeemulsification.

Oil-in-water emulsions (20 vol% oil) were prepared atroom temperature using a laboratory-scale jet homogenizerworking at the operational pressure of 350 bar (Burgaud,Dickinson, & Nelson, 1990). Emulsion droplet-size dis-tributions were measured using a Malvern MastersizerMS2000 static light-scattering analyser. The opticalabsorption parameter was set at 0.001 and refractive indexvalues of 1.330, 1.460, 1.473 and 1.470 were used for water,MCT oil, orange oil and oil mixture, respectively. Thedroplet size was characterized in terms of the averagediameter d43, defined by

d43 ¼X

inid

4i

.Xinid

3i ,

Table 1

Abbreviations used in the text to identify the protein and conjugate

emulsifier samples used in the formulation of the emulsions

Protein Abbreviation Maltodextrin

DE ¼ 2 DE ¼ 19 DE ¼ 47

MD2 MD19 MD47

Whey protein

isolate

WP WP–MD2 WP–MD19 WP–MD47

Soy protein

isolate

SP — SP–MD19 —

where ni is the number of droplets of diameter di. The d43value was used to monitor changes in the droplet-sizedistribution on storage, since this parameter was foundpreviously (Akhtar & Dickinson, 2003) to be more sensitiveto such changes than other mean droplet sizes (e.g. d32).States of droplet flocculation were assessed qualitatively byexamining emulsions by light microscopy. Creamingstability was assessed visually by determining the time-dependant thicknesses of cream and serum layers inemulsions stored quiescently at 22 1C.

3. Results and discussion

3.1. Appearance and solubility of whey protein–maltodextrin

conjugates

The dry mixture of WP and MD was white. After heattreatment at 80 1C for 2 h the mixture was of a pale goldenbrown and silky appearance, and having a pleasant smell.The most obvious immediate benefit of coupling poly-

saccharide to WP is the striking improvement in the ease ofdissolution and the solubility around pI. Conjugates werefound to dissolve immediately in citrate buffer (pH 3, ionicstrength 0.2M) to give pale brown solutions. In contrast,the original WP sample took about 2 h to dissolve into aclear solution whilst stirring at ambient temperature. Andat pH 4.7 the WP solutions were turbid, whereas theequivalent conjugate solutions remained clear throughoutthe tested pH range from 3.0 to 5.5.

3.2. SDS-PAGE analysis of whey protein–maltodextrin

conjugates

In order to establish that covalent coupling of WP toMD had indeed occurred during our relatively short dry-heating treatment times, we used SDS-polyacrylamide gelelectrophoresis (SDS-PAGE), as previously reported instudies of other protein–polysaccharide conjugate systems(Kato, Mifuru, Matsudomi, & Kobayashi, 1992; Shu,Sahara, Nakamura, & Kato, 1996). Protein componentswere identified with a Coomassie blue stain and poly-saccharide components with a PAS stain (pink). Fig. 1shows the SDS-PAGE patterns of native WP and mixturesof WP+MD (DE ¼ 19) (WP+MD19) in the weight ratio1:2 incubated at 80–100 1C for various periods of time t. Onvisualizing the gel, changes in the characteristic bands ofthe proteins were observed (Fig. 1(a)). As the conjugationreaction proceeded at 90 1C (t ¼ 1�1.5 h), a gradualdisappearance in the characteristic band pattern for theprotein could be observed. However, the appearance andposition of the protein bands did not change so signifi-cantly after dry-heating the mixture of WP–MD19 at100 1C for 1 h (see Fig. 1). The residual presence of thecharacteristic WP bands in the heated mixtures isinterpreted as being indicative of significant unreactedprotein in the conjugate sample.

ARTICLE IN PRESS

Lane 1 2 3 4 5 6 7 8 9

80°C 90°C 100°C

kDa

(a)

(b)

205

11697846655

45

36

29

24

20

Fig. 1. Analysis by SDS-PAGE of dry-heated whey protein (WP)+mal-

todextrin (MD) mixtures (weight ratio 1:2) showing effectiveness of

conjugation of WP with maltodextrin (DE ¼ 19) (MD19): (a) protein stain

(blue) and (b) carbohydrate stain (pink). The labelled lanes are: (1) MD19;

(2) native WP; (3) WP+MD19 heated at 80 1C for 2 h; (4) WP+MD19

heated at 90 1C for 1 h; (5) WP+MD19 heated at 90 1C for 1.5 h; (6)

WP+MD19 heated at 90 1 for 2 h; (7) WP+MD19 heated at 100 1C for

0.5 h; (8) WP+MD19 heated at 100 1C for 1 h; (9) WP+MD19 heated at

100 1C for 1.5 h.

0

5

10

15

20

25

d 43 (µ

m)

M. Akhtar, E. Dickinson / Food Hydrocolloids 21 (2007) 607–616610

The gel traces for the carbohydrate stain are shown inFig. 1(b). We can see that, on increasing the incubationtime from t ¼ 1 to 2 h, the pink-staining carbohydratecomponent was increasingly retained in association withthe high-molecular-weight proteinaceous material, formingcharacteristic purple-staining conjugate bands. Hence, asindicated by these new polydispersed bands present in thedry-heated mixtures, the SDS-PAGE patterns confirm thatthe WP had indeed become complexed with MD to formconjugates of higher molecular weight. The mutualconsistency of the protein bands in Fig. 1(a) with thecorresponding carbohydrate bands in Fig. 1(b) providesfurther evidence for strong (non-physical) bonding betweenprotein and the polysaccharide.

WP t=2h t=1 t=1.5 t=2h t=0. 5 t=1h t=1.5

80°C 90°C 100°C

Fig. 2. Influence of temperature/time conditions during preparation of

conjugates at relative humidity 79% on the initial average droplet size d43of emulsions (20 vol% oil mixture, pH 3.2) stabilized by 2wt%

WP–MD19 with protein/polysaccharide weight ratio 1:2. Also shown

for comparison is the initial d43 for the equivalent emulsion prepared with

the untreated pure whey protein.

3.3. Emulsifying properties of whey protein–maltodextrin

conjugates

The emulsifying efficiency of WP isolate alone, and itsconjugates with MD, has been investigated via theformulation of 20 vol% oil-in-water emulsions at pH 3.2

and 7.0 (ionic strength 0.2M). The results are comparedwith those for the reference emulsifier GA. We haveinvestigated three different samples of MD (DE ¼ 2,Mw ¼ 280 kDa; DE ¼ 19, Mw ¼ 8.7 kDa; DE ¼ 47, Mw ¼

2 kDa) at various protein/MD weight ratios, and threedifferent oil phases (MCT oil, orange oil, and oil mixture).The emulsification properties have been assessed in termsof average droplet size, creaming stability, and microscopicand visual stability observations.

3.3.1. Effects of incubation time and temperature

We have investigated the effect of thermal treatmentconditions on the emulsifying properties of the WP–MDconjugates. Samples were prepared by dry-heating ofWP+MD in the weight ratio 1:2 at 80, 90, and 100 1Cfor various incubation times. Fig. 2 shows the effect ofincubation conditions on the initial average droplet size ofoil-in-water emulsions (2wt% emulsifier, 20 vol% oilmixture) at pH 3.2. It can be seen that the conjugateWP–MD19 (weight ratio 1:2) made by dry-heating at 80 1Cfor 2 h (or 90 1C for 1.5 h) shows much better emulsifyingproperties in terms of average droplet size (d43o1 mm)when compared to the commercial WP sample alone(d43�10 mm). The emulsifying results shown in Fig. 2 forthe WP–MD19 conjugates made by dry-heating at80–100 1C for various times appear well correlated withthe extents of conjugation indicated by the SDS-PAGEpatterns (see Fig. 1).

3.3.2. Effect of the ratio of whey protein to maltodextrin

In order to determine the approximate protein–polysac-charide ratio giving optimum emulsion stability, experi-ments were carried out with various conjugates as afunction of the conjugate composition and the type of MD(DE ¼ 2, 19, and 47). Oil-in-water emulsions (2wt%emulsifier, 20 vol% orange oil) were prepared at pH 3.2.

ARTICLE IN PRESS

0

2

4

6

8

10

WP 1:1 1:2 1:3 1:4

protein/polysaccharide ratio

DE 2

DE 19

DE 47

d 43 (µ

m)

Fig. 3. Comparison of initial average droplet sizes d43 of emulsions

(20 vol% orange oil, 2 wt% emulsifier, pH 3.2) stabilized by whey protein

(WP) and WP–MD conjugates prepared from three different maltodextrin

samples (DE ¼ 2, 19, or 47) at four different protein/polysaccharide

weight ratios.

0

0.05

0.1

0.15

0.2

0.25

pH = 3.4 pH = 4.6 pH = 5.5

1:0.5

1:3

Optical density

Fig. 4. Optical densities of concentrated aqueous solutions (13wt%) of

WP–MD19 conjugates with protein/polysaccharide weight ratios of 1:0.5

and 1:3 at three different pH values.

0

3

6

9

12

(a)

(b)

15

GA WP WP−MD

orange oil

oil mixture

d 43 (µ

m)

0

1

2

3

4

5

6

GA WP WP−MD

orange oil

oil mixture

d 43 (

µm)

Fig. 5. Comparison of average droplet sizes d43 for emulsions of orange

oil or oil mixture (20 vol% oil, 2wt% emulsifier) stabilized by GA, WP or

WP–MD19 (ratio 1:2) after ambient temperature storage for 40 days at (a)

pH 3.2 and (b) pH 7.0.

M. Akhtar, E. Dickinson / Food Hydrocolloids 21 (2007) 607–616 611

Fig. 3 compares the initial droplet sizes of emulsionsstabilized by WP and WP–MD conjugates. We can see thatthe WP–MD19 samples give substantially better emulsify-ing properties in terms of average droplet size (d43o2 mm)than the WP–MD2 samples (d43�6–8 mm) under similarconditions (i.e. emulsifier concentration, pH, ionicstrength), and slightly better properties than theWP–MD47 conjugates. A possible explanation for thepoorer emulsifying characteristics of the WP–MD47conjugate is that the molecular size of the low-molecular-weight carbohydrate moiety (Mw ¼ 2 kDa) is below thatrequired for optimum steric stabilization. We also see fromFig. 3 that the mass ratios of WP to MD19 conferring thesmallest mean droplet sizes are 1:1 and 1:2. The presence ofsome unreacted protein in the 1:1 ratio conjugate may beslightly beneficial to its emulsifying capacity—although notto long-term stabilization in beverage-type formulations(see later).

In our previous emulsification work with Maillard-typecomplexes of BSA+dextran (Dickinson & Semenova,1992) and WP+dextran (Akhtar & Dickinson, 2003), theoptimum protein/polysaccharide ratio was found to bearound 1:3. However, in these previous studies thepolysaccharide moiety was of substantially higher mole-cular weight.

3.3.3. Effect of pH on solubility of WP–MD conjugates

The WP solution became turbid at pH 4.6, whereas theequivalent conjugate solutions remained clear throughoutthe pH range tested (3.2–5.5). Optical density data for someconcentrated solutions of conjugates are presented inFig. 4. For a WP–MD19 conjugate made at 1:0.5 weightratio, a slight increase in the optical density was observedat the solution concentration of 13wt% at pH 4.6. Theincrease in optical density at pH 4.6 is attributed to thepresence of some unreacted protein present in the solutionat a pH close to the protein’s isoelectric point. ForWP–MD19 conjugates of weight ratio 1:3 (or 1:5),

however, the optical density was found to remain constantfor 3.2ppHp5.5. This is consistent with there being nodetectable amount of unreacted protein present in theseconjugates, and it provides confirmation that complete andeffective coupling of the WP to the MD takes place on dry-heating these ingredients at 80 1C for a couple of hours.

3.3.4. Comparison of gum arabic and WP–MD conjugates

The ability of the conjugates to form stable oil-in-wateremulsions has been compared with that of GA under bothacidic and neutral conditions. Fig. 5 shows average dropletsizes for emulsions (20 vol% orange oil or oil mixture,

ARTICLE IN PRESS

0

20

40

60

80

100

SP SP−MD WP WP−MD

1 day

30 days

d 43 (µ

m)

0

10

20

30

40

SP SP−MD WP WP−MD

1 day

30 days

(a)

(b)

Ser

um la

yer

sepa

ratio

n (%

)

Fig. 6. Comparison of properties of emulsions (20 vol% MCT oil, 1wt%

emulsifier, pH 3.2) stabilized by pure protein (WP or SP) and conjugates

(WP–MD19 or SP–MD19) after 1 day or 30 days at ambient temperature:

(a) average droplet size d43; (b) percentage serum separation.

M. Akhtar, E. Dickinson / Food Hydrocolloids 21 (2007) 607–616612

2wt% emulsifier) stabilized by WP, WP–MD19 conjugate(1:2) and GA at pH 3.2 and 7 after a quiescent storageperiod of 40 days. The results show that the conjugate-stabilized emulsions made from the oil mixture wereextremely stable under neutral conditions. The conjugatewas found to be an especially effective emulsifier of the oilmixture, with good retention of a low mean droplet size(d43�0.5 mm) and no creaming instability observed by eyeover the experimental storage period of 40 days. At thesame emulsifier/oil ratio, the conjugates performed muchbetter than GA.

Under acidic conditions, the systems based on GA orWP exhibited poor stability in terms of retention of averagedroplet size on extended storage. The average droplet sizesafter 40 days were d43�12 mm and d43�3 mm for WP-stabilized and GA-stabilized emulsions, respectively. How-ever, emulsification with the WP–MD19 conjugate pro-duced much smaller droplets (d43�0.5 mm) than with eitherGA or WP at pH 3.2.

The good retention of the small emulsion droplets duringextended storage indicates that the WP–MD19 conjugate(weight ratio 1:2) is a highly effective stabilizer of flavouroil emulsions under these conditions.

3.4. Comparison of WP–MD conjugates with soy

protein–maltodextrin conjugates

We have investigated the effect of replacing the WP withSP isolate in these formulations. Complexes were madefrom a mixture of SP+MD19 at a protein/polysaccharideweight ratio of 1:2 under exactly the same dry-heatingconditions as used to make the WP–MD19 conjugates, andthe emulsifying properties were compared for both types ofsystems at pH 3.2 or 5.8 and ionic strength 0.2M.

Emulsifying efficiencies of pure WP, pure SP and theirrespective conjugates (WP–MD19 and SP–MD19) weretested in relation to their ability to form stable MCT oil-in-water emulsions (20 vol% oil, 1wt% emulsifier). Data foraverage droplet sizes d43 and percentage serum separationat pH 3.2 are presented in Fig. 6. We see that the initialaverage droplet sizes (measured within 1 day) weresubstantially greater for the SP-stabilized and SP–MD19-stabilized emulsions (d43�20–40 mm), with the WP–MD19-stabilized emulsions exhibiting much better storage stabi-lity than the other three tested ingredients under theseconditions. In terms of creaming stability under gravity,there was very extensive serum separation in the emulsionmade with the SP–polysaccharide conjugate, whereas theemulsion sample stabilized by the WP–MD19 conjugateexhibited relatively modest serum separation over the sameperiod of 30 days. The extensive breakdown of the SP-stabilized emulsions is due to droplet coalescence.

Therefore, we found no significant improvement inemulsification properties of SP isolate by dry-heating itwith MD under the same conditions that producedWP–MD conjugates with excellent emulsifying properties.This may be because the dry-heated vegetable protein

crosslinks faster with other protein molecules than with theMD, thereby leading to less improvement in solubility (andhence emulsifying efficiency) as a result of the dry-heatingtreatment. Anyway, the favourable comparison with SP inFig. 6 justifies the choice of the milk protein as a cheap andeffective protein ingredient for making conjugate emulsi-fiers for use in low-pH formulations.

3.4.1. Effects of elevated temperature and electrolyte

addition on emulsion storage stability

Tests were carried out to determine the electrolytetolerance and temperature stability of the WP–MD19-stabilized conjugate emulsions. Again the main referenceemulsifier was GA, and data were obtained also for WP, SPand SP–MD19. In these tests, the emulsions were madewith MCT oil or orange oil (20 vol%) at a total emulsifierconcentration of just 1wt% in the presence and absence ofSL at pH 5.8. The emulsions without SL were heatedwithout disturbance in a water bath at a constanttemperature of 40 1C for 3 weeks.Fig. 7(a) compares average droplet sizes of emulsions

stored in the presence of SL. We can see that, in terms ofelectrolyte tolerance, the WP–MD19 conjugate performsvery favourably in the MCT oil-in-water emulsions, ascompared with the other four tested ingredients. Thecorresponding results for the elevated temperature storagetest are presented in Fig. 7(b). Again, the ability of the

ARTICLE IN PRESS

0

15

30

45

60

GA SP SP−MD WP WP−MD

1 day

21 days

d 43 (µ

m)

d 43 (µ

m)

0

15

30

45

60

GA SP SP−MD WP WP−MD

1 day

21 days

(a)

(b)

Fig. 7. Comparison of average droplet sizes d43 of emulsions (20 vol%

MCT oil, 1wt% emulsifier, pH 5.8) stabilized by GA, WP, SP, WP–MD19

and SP–MD19 after 1 day or 21 days: (a) with added sodium lactate

(5 wt%); (b) stored at elevated temperature (40 1C).

0

9

18

27

36

45

3.5 4.0 4.5 5.0pH

WP-MD19 (1:3)

WP-MD19 (1:1)

WP-MD19 (1:0.5)

d 43 (µ

m)

0

3

6

9

12

15

WP 1:1 1:2 1:3 GAprotein/polysaccharide ratio

Original Emulsion

Coloured Emulsion

d 43 (µ

m)

(a)

(b)

Fig. 8. Stability of concentrated coloured emulsions (20 vol% oil mixture,

2wt% emulsifier): (a) Effect of pH on average droplet size d43 of coloured

emulsions stabilized by WP–MD19 conjugates of protein/polysaccharide

weight ratios 1:0.5, 1:1 and 1:3 after storage at ambient temperature for 8

days. (b) Comparison of average droplet sizes d43 of original (white) and

coloured emulsions (pH 3.2) stabilized by GA, WP and WP–MD19

conjugates of ratios 1:1, 1:2 and 1:3 after storage at ambient temperature

for 13 days.

M. Akhtar, E. Dickinson / Food Hydrocolloids 21 (2007) 607–616 613

WP–MD19 conjugate to confer excellent storage stabilityon these emulsion systems is clearly demonstrated.

3.5. Properties of emulsions containing colouring agents

3.5.1. Stability of concentrated coloured emulsions

In order to improve the appearance of beverageformulations, additional colouring materials are commonlyincorporated (Garti & Pinthus, 1998; Tan, 1997). Further-more, the cloudy appearance is an important property ofcitrus soft drinks (orange, lime, lemon) because it gives thenatural look of real fruit juice (Dickinson, 1994). Theformulated beverage is typically made by diluting aconcentrated cloudy emulsion in a sugar solution; thesystem in both the concentrated and the diluted emulsionforms must be highly stable, with the industry normallyrequiring a shelf-life of at least 6 months (Tan, 1997). Inorder to match this stringent stability requirement, themean droplet size of the emulsions should be as small aspossible, but when it is below 0.5 mm the cloud intensitymay be reduced (Dickinson, 1994; Garti & Pinthus, 1998).

Here, first, we test the stability of the concentratedcoloured emulsion system made with WP–MD (DE ¼ 19)conjugates. Some samples of orange-coloured emulsionwere prepared by mixing in the ratio 70:30 (by volume) a

normal (whitish) emulsion (2.5wt% WP–MD19, 20 vol%oil mixture) and a coloured solution containing sodiumbenzoate, citric acid and the colouring agents. The effect ofthe colourant on the stability of emulsions of various pHvalues, ionic strengths, and WP/MD ratios was theninvestigated. The original non-coloured emulsions(20 vol% oil) were prepared at various pH values andsubsequently were mixed with the coloured solution.Upon mixing with the coloured solution, the emulsion

stability properties appeared to become more sensitive topH. Droplet sizes of WP–MD19-stabilized emulsions madeat low pH, after storage for 8 days, were found to bedependant on the protein–polysaccharide ratio, as shownin Fig. 8(a). The emulsion made at pH 3.5 with WP–MD19(ratio 1:0.5), when mixed with the coloured solution,exhibited flocculation/ precipitation after 24 h. On increas-ing the pH of the emulsion to pH 4.5 and mixing with thecoloured solution, the resulting system appeared rathermore stable. More strikingly, however, much greaterstability was achieved by increasing the protein–polysac-charide ratio to 1:1 or 1:3; in particular, coloured

ARTICLE IN PRESS

Fig. 9. Photographs of tubes containing samples of concentrated and

extensively diluted emulsions after storage for 10 days at ambient

temperature: (a) The concentrated emulsions (20 vol% oil mixture, 2wt%

emulsifier, pH 3.2, ionic strength 0.2M) stabilized byWP (A) andWP–MD19

conjugates of protein/polysaccharide weight ratios 1:1 (B), 1:2 (C) and 1:3

(D). (b) The same emulsions diluted 1:1000 by volume according to the

procedure described in the text. Sample (A) has a precipitate (P) at the

bottom of the tube. Pictured also is a reference sample (GA) made by diluting

a gum arabic-stabilized concentrated emulsion (20 vol% oil mixture, 30wt%

emulsifier). (We note that although these photographs were actually taken

after 10 days, no further discernible change in the visual appearance of the

tubes could be detected after 30 days.)

M. Akhtar, E. Dickinson / Food Hydrocolloids 21 (2007) 607–616614

emulsions containing WP–MD19 (ratio 1:3) were found toexhibit excellent stability in terms of maintenance of a lowmean droplet size over the whole pH range 3.5–5.5.

Table 2 and Fig. 8(b) present a comparison of theaverage droplet sizes of the original and coloured emul-sions prepared with WP, GA and WP–MD19 conjugates of1:1, 1:2 and 1:3 protein/polysaccharide ratio and thenstored at ambient temperature for 13 days. A minimalchange in mean droplet sizes was observed for the ratios 1:2or 1:3 upon mixing with colourant. Since it is the free(uncomplexed) protein that is considered to be highlysusceptible to precipitation at low pH in the presence of thecolourant, these data provide further confirmation thatessentially all the protein was effectively conjugated withpolysaccharide when dry-heated at a weight ratio of 1:2 orhigher. It is noteworthy from Fig. 8(b) that, at the ratio of2.5wt% emulsifier to 20 vol% oil, the WP–MD conjugateis much more effective in producing stable colouredemulsions than is GA. Nonetheless, it should be notedthat, as expected from the literature, the GA can make finestable coloured emulsions at the high ratio of 20wt%emulsifier to 20 vol% oil (see Table 2).

Fig. 9(a) shows the visual appearance of concentratedcoloured emulsions prepared at pH 3.2 (ionic strength0.2M) and stored at ambient temperature for 10 days. Thephotographs show that coloured emulsions stabilized bythe WP–MD19 conjugates of 1:2 or 1:3 ratio have excellentstability with respect to flocculation/precipitation whencompared with the WP-stabilized emulsion. In fact, it wasobserved that the conjugate-stabilized coloured emulsionshave no discernible phase separation after 30 days. Theseresults for the concentrated low-pH coloured emulsionsshow that the conjugate composition favouring the lowestinitial droplet size is not necessarily the one recommendedas the optimum protein–polysaccharide ratio for beverageformulations. That is, although a low protein/polysacchar-ide ratio (1:1) is perhaps most favourable in terms of theinitial mean droplet size (see Fig. 3), the ratio has to beincreased slightly to 1:2 or 1:3 in order to reduce tonegligible proportions the level of unreacted protein, andso to eliminate the possibility of any potential instabilitylater caused by aggregation of unreacted protein on mixingwith the colouring agent.

Table 2

Average droplet size d43 and visual appearance of original emulsions (20 vol% oil mixture) and concentrated coloured emulsions (obtained by mixing 70:30

with colouring agent) stabilized by WP, WP–MD19 and GA at pH 3.2 after a storage period of 13 days

Emulsifier Aqueous phase content

before emulsification (wt%)

Droplet size d43 (mm) Appearance of coloured emulsion

Original emulsion Coloured emulsion

WP 2.5 6.6 32 Phase separation, precipitation

WP–MD19 (2:1) 2.5 — — Phase separation, precipitation

WP–MD19 (1:1) 2.5 0.58 1.02 Uniform sample

WP–MD19 (1:2) 2.5 0.55 0.75 Uniform sample

WP–MD19 (1:3) 2.5 0.77 0.97 Uniform sample

WP–MD19 (1:4) 2.5 0.85 1.41 Uniform sample

GA 30 0.65 0.68 Uniform sample

ARTICLE IN PRESSM. Akhtar, E. Dickinson / Food Hydrocolloids 21 (2007) 607–616 615

3.5.2. Stability of diluted coloured emulsions

The stability of the coloured emulsions was testedfurther on dilution with sugar syrup to achieve acomposition representative of a carbonated soft drinkapplication. The system was prepared by diluting 3 g of20% oil-in-water coloured emulsion in 500ml of aqueoussugar syrup (containing sodium benzoate and citric acid).One part of the sugar syrup was diluted with five parts ofcarbonated water. Systems containing conjugates madefrom various ratios of WP and MD19 were compared withthose based on GA present at the high emulsifier level(30%) commonly encountered in commercial flavour oilemulsion formulations.

Coloured emulsions stabilized by WP–MD19 of protein/MD ratio 1:2 or 1:3 were found to be completely stabletowards extensive dilution at pH ¼ 3.2. As shown in thephotograph in Fig. 9(b), there was no flocculation,precipitation or phase separation apparent over a storageperiod of 10 days at ambient temperature. (The appearanceof the sample tubes remained unchanged after 1 month ofstorage.) In contrast, the equivalent diluted emulsionobtained with the protein used alone as emulsifier gave acoloured precipitate at the bottom of the tube. By eye, ourconjugate-stabilized beverage-type emulsions were identicalin appearance to the reference emulsions stabilized by GA.

4. Conclusions

Covalent protein–polysaccharide complexes made bymoderately short dry-heating treatments of mixtures ofWP+MD have functional properties that offer substantialpotential for use as cheap and effective food ingredients. Inparticular, these complexes have excellent solubility andexceptional emulsification properties under acidic condi-tions. The emulsifying and emulsion stabilizing propertiesof the WP–MD conjugates are effective at low emulsifier/oil ratio, and under aqueous conditions of high electrolyteconcentration (5% SL) or elevated storage temperature(40 1C). Furthermore, this physical stability is maintainedin the presence of food colouring agents, both before andafter extensive dilution.

It has been demonstrated therefore that a conjugatebased on a mixture of WP and a MD of intermediatemolecular size (�10 kDa) is capable of producing fineemulsion droplets (0.5–1 mm) with either triglyceride oil ororange oil. The conjugate WP–MD19 can be used as aneffective emulsifier for formulating food emulsions underacidic conditions and at high salt concentrations. Inparticular, triglyceride oil-in-water emulsions made withWP–MD19 at relatively low conjugate/oil ratios have beenfound to have excellent stability behaviour in terms ofaverage droplet size and creaming behaviour over a3-month storage period.

The primary positive effect of WP conjugation with MDis the greatly improved solubility at low pH. This enhancedsolubility is largely responsible for the much betteremulsifying properties of the WP–MD conjugate as

compared with the protein alone. The improved emulsify-ing properties are attributable to the enhanced stericstabilization provided by the bulky hydrophilic polysac-charide moiety, as has been discussed previously elsewhere(Akhtar & Dickinson, 2003; Dickinson, 1995; Dickinson &Galazka, 1991).A major potential application of this type of protein–

polysaccharide complex is in the stabilization of citrus oilemulsions as an alternative to GA. We have demonstratedhere that concentrated orange oil-in-water emulsionsstabilized by WP–MD19 conjugates of 1:2 or 1:3 ratio doindeed have excellent stability in terms of the absence ofprecipitation, flocculation or phase separation upon mixingwith colouring material over the pH range 3.2–5.5. Inaddition to the extended shelf-life of the concentrates, ithas been shown that these systems can be successfullydiluted with carbonated sugar syrup to produce stabledilute coloured emulsions, with direct relevance forcommercial soft drink applications.

Acknowledgments

We acknowledge the financial support from Uniqema(ICI) and useful discussions with Trevor Blease, JackBurger and Simon Davies.

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