emulsifying properties of whey protein–dextran conjugates at low ph and different salt...
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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
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doi:10.1016/S0927-7765(03)00049-3
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.
M. Akhtar, E. Dickinson / Colloids and Surfaces B: Biointerfaces 31 (2003) 125�/132128
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.
M. Akhtar, E. Dickinson / Colloids and Surfaces B: Biointerfaces 31 (2003) 125�/132 129
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.
M. Akhtar, E. Dickinson / Colloids and Surfaces B: Biointerfaces 31 (2003) 125�/132130
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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M. Akhtar, E. Dickinson / Colloids and Surfaces B: Biointerfaces 31 (2003) 125�/132 131
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M. Akhtar, E. Dickinson / Colloids and Surfaces B: Biointerfaces 31 (2003) 125�/132132