Emulsifying properties of whey protein–dextran conjugates at low pH and different salt...

Emulsifying properties of whey protein�/dextran conjugates atlow pH and different salt concentrations

Mahmood Akhtar *, Eric Dickinson

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

Received 21 August 2002; received in revised form 1 October 2002; accepted 26 November 2002

Abstract

The emulsifying behaviour of glyco-protein complexes of the non-ionic polysaccharide dextran (500 kDa) with whey

protein isolate (WPI) have been investigated in systems containing (20 vol.% oil phase) medium-chain triglyceride oil,

silicone oil, orange oil, and n -tetradecane under acidic and high electrolyte concentrations. Covalent coupling of

protein to polysaccharide is achieved by dry heat treatment of protein�/polysaccharide mixtures. Emulsions were made

with WPI and whey protein isolate-dextran (WPI-DX) conjugate, and stability was followed by determining changes in

average droplet size and extent of serum separation with time, with gum arabic (GA) chosen as reference emulsifier. The

results show that the WPI-DX conjugate gives much better stability than the whey protein alone or GA under similar

conditions. The improved emulsifying properties of WPI on complexing with dextran is probably due to the enhanced

steric stabilization provided by the bulky hydrophilic polysaccharide moiety.

# 2003 Elsevier B.V. All rights reserved.

Keywords: Whey protein; Protein�/polysaccharide complex; Emulsion stability; Dextran; Droplet size

1. Introduction

Both protein and polysaccharide play promi-

nent role in the formulation of food emulsions.

Proteins act as emulsifiers and stabilizers of

emulsion droplets against aggregation and coales-

cence. Stability of these emulsions can be enhanced

by using high-molecular weight-polysaccharides

that keep droplets apart after their formation

and thus protect them against creaming, floccula-

tion and coalescence [1]. The functional properties

of proteins can be further improved by linkage

with polysaccharides. The high-molecular-weight

glyco-conjugate is supposed to combine the prop-

erties of a hydrophobic protein, being firmly

attached to the oil droplet surface, with the

property of a hydrophilic polysaccharide, being

highly solvated by the aqueous phase medium [2].

The application of a non-toxic chemical modifica-

tion to form such a conjugate is of a great

potential interest for the food industry. The

naturally occurring Maillard reaction can attach

a protein to a polysaccharide. Conjugation by the

dry heating method is based on the Amadori

rearrangement steps in the Maillard reaction

* Corresponding author. Tel.: �/44-113-343-2970; fax: �/44-

113-343-2982.

E-mail address: [email protected] (M. Akhtar).

Colloids and Surfaces B: Biointerfaces 31 (2003) 125�/132

www.elsevier.com/locate/colsurfb

0927-7765/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0927-7765(03)00049-3

mailto:[email protected]

where terminal and intrastitial amines of theprotein are linked to the reducing end of the

polysaccharide [3].

Glycosylation of b-lactoglobulin (b-LG) with

different sugars can induce a modification of the

solubility profile shifting the minimum in solubi-

lity towards more acidic pH. The modified protein

shows better thermal stability at acidic pH and

improved emulsifying properties [4]. Stability toheat treatment, as required for pasteurization and

homogenizing during emulsion formation, there-

fore produces a casein�/polysaccharide conjugate

that is superior to unmodified casein as an

industrial emulsifier [5]. Possibly it is not just

enhanced steric stabilization or polysaccharide

entanglements that are responsible for the im-

proved emulsification properties. Conjugation ofpolysaccharide to protein can also greatly improve

the solubility of the biopolymer emulsifier at acidic

pH [3].

Nagasawa et al. [6] have reported that the

emulsifying ability of b-LG can be improved by

conjugation with carboxymethyldextran (CMD) at

acidic pH, in the presence of salt and at elevated

temperature. The b-LG-CMD conjugates ofhigher CMD content showed better emulsifying

properties than those with low CMD content

under these unfavorable solution conditions. How-

ever, there has so far been little research on

improving the emulsifying properties of commer-

cial whey protein isolate (WPI) with non-ionic

(unmodified) dextran (DX) under similar acidic

conditions and high salt concentrations.We report here on preparation of covalently

linked whey protein isolate-dextran (WPI-DX)

complexes and formulation of fine glycoprotein-

stabilized oil-in-water (O/W) emulsions using low

concentrations of the glycoprotein. Our objective

is to evaluate the emulsifying properties of the

WPI�/DX conjugates, as compared with those for

the commercial sample of WPI under acidic andhigh salt concentration. These conditions of low

pH and high ionic strength are often found in food

systems. The neutral, branched polysaccharide

dextran is chosen to avoid the complications of

electrostatic complexes which often occur in mix-

tures of proteins with anionic polysaccharides

[7,8]. Gum arabic (GA) is chosen as a reference

emulsifier because of its common use in beverage

emulsions under acidic conditions. The globular

protein (whey protein) is chosen in an attempt to

improve its emulsifying properties through the

enhanced steric stabilization provided by linkage

to a bulky hydrophilic polysaccharide moiety. The

protein�/polysaccharide complexes are tested in

emulsion formulations under acidic and concen-

trated electrolyte conditions. The effect of the

molar ratio R of protein to polysaccharide on

the emulsion stability is investigated for the WPI-

DX conjugate. All emulsions contain the same

amount of total emulsifier (for conjugate, 0.8 wt.%

protein�/2.4 wt.% DX) and oil (20 vol.%), and the

results are compared with those for emulsions

containing 3.2 wt.% GA (as reference emulsifier).

The conjugates are ‘natural’ and non-toxic, and

with their improved emulsifying properties in the

presence of acids and salts, have significant

potential for use in the food and personal care

industries.

2. Materials and methods

2.1. Materials

Whey Protein Isolate, BiPro (WPI), was ob-

tained from Davisco Food International. It was

manufactured from fresh, sweet dairy whey that

was concentrated and spray dried. The product

was in the form of a homogenous, semi-hygro-

scopic lactose-free powder. The medium-chain

triglyceride oil (MCT oil) and the flavouring oil

(orange oil) were provided by Quest International.

Polysaccharide dextran (average molecular weight

488 kDa, D-1037, Lot 72H0717) and b-LG from

bovine milk (:/90% (PAGE), L-3908, Lot

101K7031) were purchased from Sigma Chemi-

cals, as was the AnalaR grade n -tetradecane.

Silicone oil (Dow Corning 200/20cS fluid, Lot

061601) and sodium lactate solution (�/50%, Lot

K24888657) were purchased from BDH Labora-

tory Supplies.

M. Akhtar, E. Dickinson / Colloids and Surfaces B: Biointerfaces 31 (2003) 125�/132126

2.2. Coupling of protein and polysaccharide

Protein and polysaccharide were brought into

good contact by dissolving the WPI and DX in

distilled water in the weight ratio of 1:3. The

samples were then freeze-dried to remove the

water, and were ground to make the powder. A

desiccator was placed in the oven at 80 8C for 10�/

15 min to achieve the equilibrium temperature.Then the sample was placed in the desiccator for 2

h for the conjugation. After incubation at 80 8Cfor 2 h, a light brownish colour appeared due to

non-enzymatic browning at relative humidity of

79%. The incubation temperature could be re-

duced by increasing the incubation time. In addi-

tion to 1:3 weight ratio, conjugates of various ratio

of WPI-DX were also made. The resultant glyco-conjugate was analysed using the SDS-PAGE

technique to establish the presence of protein�/

polysaccharide conjugation. The efficiency of the

conjugates to stabilize 20 vol.% O/W emulsions

was then tested.

2.3. SDS-Polyacrylamide Gel Electrophoresis

SDS-Polyacrylamide Gel Electrophoresis (SDS-

PAGE) was performed according to the method of

Laemmli [9] using a 14% acrylamide separating gel

and a 6% stacking gel containing 0.1% SDS.

Samples (30 ml, 0.1% of protein) were prepared

in a 0.01 M Tris�/glycine buffer at pH 8.8 contain-

ing 1% SDS. Electrophoresis was carried out at a

constant current of 15 mA on a gel for 2 h in aTris�/glycine buffer containing 0.1% SDS. The gel

sheets were stained for protein (0.2% Coomassie

brilliant blue G-250) and carbohydrate (0.5%

periodate-fuchin solution) [10].

2.4. Emulsion preparation

The aqueous buffer (ionic strength 0.2 M) was

prepared using citric acid, sodium citrate anddouble-distilled water, with 0.01 wt.% sodium

azide added as antimicrobial agent. Protein or

protein/polysaccharide conjugate was added

slowly to the buffer solution at ca. 22 8C with

gentle stirring. The pH of the resulting protein

solution was adjusted to pH 3.2 by adding a few

drops of 1 M HCl. Subsequently reported pHvalues refer to the pH of the protein solution

before emulsification.

O/W emulsions (20 vol.% oil) were prepared at

room temperature using a laboratory-scale jet

homogenizer working at the operational pressure

of 300 bar [11]. Emulsion droplet-size distributions

were measured using a Malvern Mastersizer

MS2000 static laser light-scattering analyser withabsorption parameter value of 0.001. The average

droplet size was characterized by two mean

diameters, d32 and d43, defined by

d32�Sinid3i =Sinid

2i ; d43�Sinid

4i =Sinid

3i ;

where ni is the number of droplets of diameter di .

The d32 value was used to estimate the specific

surface area of freshly made emulsions, and the d43

value was used to monitor changes in droplet-size

distribution on storage. States of droplet floccula-

tion were assessed qualitatively by examining

emulsions by light microscopy using the Nor-

marski differential interference contrast technique

[12]. Creaming stability was assessed visually by

determining the time-dependent thicknesses of

cream and serum layers in emulsions storedquiescently at 22 8C.

3. Results and discussion

3.1. SDS-PAGE analysis

SDS-PAGE was used to establish the covalent

coupling of the whey protein to the DX after dry-heat treatment. Fig. 1 shows the SDS-PAGE

patterns of native WPI, pure b-LG, and putative

whey protein�/DX (Mw 488 kDa) conjugates. On

visualizing the gel, the characteristic protein bands

were visible. For the conjugates, a shift in the band

pattern for the protein was observed (compare the

bands in lanes 2 and 4). The characteristic bands

of the protein alone (in the conjugate sample) havenot fully disappeared, indicating that not all the

protein has reacted with the polysaccharide under

the heating conditions employed. Probably this is

due in part to the molecular weight of the

polysaccharide used*/a small molecule DX might

bind more rapidly to protein to produce surface-

M. Akhtar, E. Dickinson / Colloids and Surfaces B: Biointerfaces 31 (2003) 125�/132 127

active complex than is possible with the morebulky DX molecules.

The SDS-PAGE patterns show polydispersed

bands at the top of the separating gel in the

conjugate suggesting the formation of high mole-

cular weight products. The positions of the poly-

dispersed protein bands (blue colour) are

consistent with carbohydrate bands (pink colour),

demonstrating the covalent linkage between pro-tein and the polysaccharide. For other systems

(e.g. casein�/DX and lysozyme�/galactomannan

conjugates), the formation of a covalent linkage

on dry-heating of protein and polysaccharide has

been verified using SDS-PAGE analysis [5,13].

3.2. Emulsion stability

A typical scan of the initial average droplet size

profiles of whey protein-stabilized emulsions as a

function of emulsifier concentration is shown in

Fig. 2. As expected, increasing the concentrationof whey protein decreases the average droplet size.

At the concentration of 1.5 wt.%, fine O/W

emulsions of MCT oil can be made with average

droplet sizes of d32�/0.4 mm and d43�/1 mm at pH

3.2 and ionic strength 0.2 M. On the basis of this

scan, the concentration of whey protein was

chosen thereafter to be 0.8 wt.% for making the

protein�/polysaccharide complexes. The 0.8 wt.%

whey protein-stabilized emulsions have a relatively

large droplet size (d43�/3 mm) under the acidic

conditions employed, and thus there is scope for

substantial improvement on coupling of the pro-

tein to the DX.

In order to choose an effective protein�/poly-

saccharide ratio to give optimum emulsion stabi-

lity, various WPI-DX ratios have been tested in

the formulation of MCT O/W emulsions. Emul-

sion stability was assessed over a period of 4�/12

weeks on the basis of droplet-size measurement,

creaming behaviour, and light microscopy obser-

vations. Fig. 3 compares the average emulsion

Fig. 1. SDS-PAGE of whey protein and whey protein�/DX

conjugate. (A) Protein stain; Lane 1, marker proteins; Lane 2,

WPI; Lane 3, b-LG; Lane 4, WPI-DX conjugate; (B) carbohy-

drate stain for WPI-DX conjugate. Arrow indicates the

boundary between stacking and separating gels.

Fig. 2. Effect of whey protein (WPI) concentration on the

initial average droplet sizes of whey protein-stabilized emulsion

of MCT O/W emulsions (20 vol.%) at pH 3.2, ionic strength 0.2

M.

Fig. 3. The influence of protein:polysaccharide molar ratio (R )

on emulsifying capacity of WPI-DX conjugate for MCT O/W

emulsions (20 vol.%) made with 3.2 wt.% total emulsifier

concentration at pH 3.2, ionic strength 0.2 M.


droplet size d43 against molar ratio R for WPI-DXconjugates over a storage period of 31 days at pH

3.2. The (1:3) WPI-DX conjugate was capable of

producing small emulsion droplets and serum

separation was not noticeable over a storage

period of 2 months. We see from Fig. 3 that the

molar ratio of WPI to DX conferring the best

emulsion stability is R :/7. This corresponds to

the weight ratio of protein to polysaccharide 1:3.In our previous work [7] on Maillard complexes of

BSA with DX, the complexes were prepared with a

protein to polysaccharide weight ratio of 1:3,

which is similar to that studied here. The con-

jugate-stabilized emulsions have shown better

emulsifying properties in terms of average droplet

size (d43B/0.5 mm) and creaming behaviour when

compared to emulsions made with the commercialwhey protein alone (d43�/3 mm). The weight ratio

of WPI to DX (molecular weight 500 kDa) for the

best emulsion stability was found to be 1:3, and

this ratio was therefore chosen for the rest of the

experiments described in this paper.

The conjugate emulsifier is not a single species,

and it contains some unreacted protein. It is

possible that some of the unreacted protein couldalso coexist with the complex in the adsorbed layer

around the droplets. However, the main contribu-

tion to the improved long term stability is the

polysaccharide of the conjugate providing a more

bulky steric stabilizing layer around the droplets.

Based on our previous work with mixed protein

layers, it seems unlikely that exchange between

unreacted protein and complex would occur be-tween the interface and bulk during long-term

storage.

The efficiency of the protein and the protein�/

polysaccharide complexes in relation to their

ability to form stable O/W emulsion is compared

with that of GA as reference emulsifier. Fig. 4

gives data for MCT O/W emulsions stabilized by

GA, WPI, and WPI-DX conjugate at pH 3.2. Boththe GA and the WPI systems exhibited poor

stability under acidic conditions in terms of

retention of average droplet size on extended

storage. The average droplet sizes after 800 h

were d43�/14 mm and d43�/3 mm for the WPI- and

GA-stabilized emulsions, respectively. Droplet

sizes of emulsion made with WPI alone were

found to increase steadily over the storage period

of 34 days. However, emulsification with the WPI-

DX conjugate generates much smaller droplets

(d43B/0.5 mm) than with either GA or with protein

alone at pH 3.2. The retention of a constant low

droplet size during extended storage indicates that

the WPI-DX conjugates are extremely efficient in

producing stable emulsions under these condi-

tions.

Emulsifying properties of WPI and WPI�/DX

conjugate have been investigated in the formula-

tion of various O/W emulsions over a storage

period of 90 days under the same acidic condi-

Fig. 4. Comparison of average droplet sizes of MCT O/W

emulsion (20 vol.%, 0.8 wt.% protein) stabilized by various

emulsifiers (3.2 wt.% total emulsifier), GA, whey protein (WPI)

or WPI-DX conjugate at pH 3.2, ionic strength 0.2 M: (a) GA;

(b) WPI alone; (c) WPI-DX conjugate.


tions. As before, results are compared with GA,

the reference emulsifier. The summary of results is

presented in Fig. 5, average droplet size after 1 day

and creaming stability over 90 days storage period.

All the WPI-DX conjugate-stabilized emulsions

were stable in terms of average droplet size and

creaming behaviour over the extended storage

period. The initial average droplet sizes of emul-

sions made with MCT oil, silicone oil and n-

tetradecane stabilized by WPI-DX conjugate were

much smaller (d43B/0.5 mm) than the equivalent

WPI-stabilized emulsion (d43�/4 mm). In terms of

creaming stability, there was very extensive serum

separation exhibited by emulsions made with WPI

alone, whereas the emulsions stabilized by WPI-DX conjugate exhibited negligible serum separa-

tion upon storage of up to 3 months (see Fig. 5(b)).

There was found to be a good correlation between

average droplet sizes and creaming stability of the

conjugate-stabilized emulsions. The extent of

serum separation after 3 months was negligible

for the conjugate-stabilized emulsions. However,

the conjugate-stabilized emulsions of the flavour-ing oil (orange oil) have shown relatively large

droplet sizes (d43�/4 mm) and poor creaming

stability on extended storage period.

As expected the conjugates were very effective

emulsifiers of the MCT oil, silicone oil and n -

tetradecane oil, with good retention of a low initial

droplet size (d43�/0.4 mm) and good creaming

stability on extended storage. These conjugatesperformed much better than the reference emulsi-

fier (GA) or WPI alone under the acidic condi-

tions.

Fig. 6 shows the stability of emulsions contain-

ing added electrolyte (5% sodium lactate). The

time-dependent average droplet sizes of MCT oil

emulsions stabilized by GA, WPI alone and the

WPI-DX conjugate at pH 3.2 are plotted over astorage period of 37 days. It has been demon-

strated clearly that the conjugates are highly

effective in stabilizing O/W emulsions in terms of

average droplet size (d43B/0.6�/0.8 mm) under

these acidic conditions in the presence of sodium

lactate over the extended storage period. Signifi-

cantly, the large initial droplet sizes (d43�/8�/36

mm) over a storage period of 37 days indicate thatboth the WPI and the GA have poor emulsifying

properties in the presence of electrolyte. A large

increase in the average droplet size is due to the

coalescence of the emulsion droplets as visualized

by light microscopy. On the basis of the results in

Figs. 5 and 6, it is apparent that the WPI-DX

complex has excellent emulsifying capabilities for

MCT oil emulsion at pH 3.2 and an impressiveionic strength tolerance.

4. Conclusions

It has been demonstrated that a WPI�/DX

conjugate is capable of producing fine emulsion

Fig. 5. Comparison of average droplet sizes and creaming

profiles of various O/W emulsions (20 vol.%) stabilized by (3.2

wt.% total emulsifier) GA, whey protein (WPI) alone and WPI-

DX conjugate at pH 3.2: (a) average droplet size after 1 day

storage; (b) creaming (as % serum layer) after storage of 90

days.


droplets and so can be used as an effective

emulsifying agent for formulating food O/W

emulsions under acidic conditions, even at high

salt concentrations. In particular, MCT O/W

emulsions made with WPI-DX conjugate at rela-

tively low conjugate/oil ratios were found to have

excellent stability in terms of average droplet size

and creaming behaviour over a 3-month storageperiod.

Based on the combined creaming stability and

time-dependent droplet size measurements in Fig.

5, we infer that the WPI-DX conjugate is an

effective stabilizing biopolymer of MCT oil, sili-

cone oil or n -tetradecane oil at pH 3.2. In addition

to the improved solubility at acidic pH, the reason

for the improved emulsifying properties of wheyprotein on complexing with polysaccharide is

presumably the enhanced steric stabilization pro-

vided by the bulky hydrophilic polysaccharide

moiety. We hypothesize that the protein moiety

of the conjugate anchors the molecule at the oil�/

water interface, and the covalently linked poly-

saccharide chain then provides a sterically stabiliz-

ing layer around the droplets which protectsagainst flocculation under conditions where elec-

trostatic stabilization is unfavourable [14,15].

Acknowledgements

We acknowledge financial support from Uni-

qema (ICI) for this project.

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Fig. 6. Influence of electrolyte (5% sodium lactate) on the

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stabilized at pH 3.2 by various emulsifiers (3.2 wt.% total

emulsifier): (a) GA; (b) whey protein (WPI); or (c) WPI-DX

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Emulsifying properties of whey protein–dextran conjugates at low pH and different salt...

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