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Characterization of Rheological and Molecular Properties of Whey

Protein Thickeners

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DOI: 10.1080/10942912.2011.642445

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Characterization of Rheological and

Molecular Properties of Whey Protein
Thickeners
a a a
Ahmed S. Eissa , Dania M. Mohamed , Kawther S. Uoness ,
a a a
Mohammed M. Azab , Noha S. Abed & Duha Abu El-Aish
a
Department of Chemical Engineering , Cairo University , Cairo ,
Egypt
Accepted author version posted online: 13 May 2013.Published
online: 08 Nov 2013.

To cite this article: Ahmed S. Eissa , Dania M. Mohamed , Kawther S. Uoness , Mohammed M.
Azab , Noha S. Abed & Duha Abu El-Aish (2014) Characterization of Rheological and Molecular
Properties of Whey Protein Thickeners, International Journal of Food Properties, 17:3, 570-586, DOI:
10.1080/10942912.2011.642445

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 International Journal of Food Properties, 17:570–586, 2014
Copyright © Taylor & Francis Group, LLC
ISSN: 1094-2912 print / 1532-2386 online
DOI: 10.1080/10942912.2011.642445

CHARACTERIZATION OF RHEOLOGICAL
AND MOLECULAR PROPERTIES OF WHEY
PROTEIN THICKENERS

Ahmed S. Eissa, Dania M. Mohamed, Kawther S. Uoness,

Mohammed M. Azab, Noha S. Abed, and Duha Abu El-Aish
Downloaded by [University of Nebraska, Lincoln] at 13:56 14 October 2014

Department of Chemical Engineering, Cairo University, Cairo, Egypt

The production of whey protein thickeners by drying protein gels to produce instant
thickening powders has been previously investigated. The use of native whey proteins as a
food thickener, however, has not been studied earlier. In this work, we characterized thermally
treated whey protein solutions with improved thickening properties. Viscosity contour maps
were constructed to outline the viscosity values at different protein and salt concentra-
tions. Huggins-Kraemer constants revealed that at low ionic strengths, polymerized protein
molecules behave as flexible coils, while at higher ionic strengths, molecules appear to be
aggregated in a spherical like shape.

Keywords: Thickener, Whey protein isolate, Viscosity, Gel point, Intrinsic viscosity, SDS-
PAGE, Huggins and Kraemer.

INTRODUCTION
The food industry’s growing demand for functionally superior and nutritionally ben-
eficial proteins provides an opportunity for increasing the utilization of whey proteins in
formulated food products. Whey proteins play an important role in nutrition as a rich
and balanced source of amino acids.[1] Moreover, they possess specific physiological
actions.[2,3] Beta-lactoglobulin (β-LG), the main constituent of whey proteins, account-
ing for about half of the protein in bovine whey isolate[4] is a rich source of cysteine,[5] one
of the most essential amino acids.
Whey proteins possess important functional properties, such as emulsification, stabi-
lization, thickening, improved appearance, taste or texture, and binding of fat or water.[6–8]
The ability of whey proteins to perform such functionalities stems from their amphiphilic
nature and conformational properties. Improvements in functional properties may be
achieved by manipulating protein conformational properties and molecular weight. β-LG
undergoes polymerization via disulphide interchange reaction to form a linear polymer
when heated at a temperature greater than protein’s denaturation temperature.[7,8] The
molecular size of β-LG does not only depend on the extent of chemical bonding, but it also
depends on the hydrogen bonding, electrostatic interactions, and hydrophobic interactions.

Received 30 August 2011; accepted 28 October 2011.

Address correspondence to Ahmed S. Eissa, Department of Chemical Engineering, Cairo University,
El-Gamaa Street, Cairo, Giza 12613, Egypt. E-mail: aseissa@gmail.com

570
 CHARACTERIZATION OF WHEY PROTEIN THICKENERS 571

Whey proteins have received little attention as food thickeners.[9] The pioneer work
that aimed at obtaining whey proteins as an instant viscofying powder is credited to Holst
et al.,[10] Thomsen,[11] and Elofsson et al.[12] who prepared the powder by heat treat-
ment during homogenization of a whey concentrate at slightly alkaline pH followed by
immediate drying.
More recent work has been done by Hudson et al.,[13] Resch and Daubert,[14] Resch
[15]
et al., and Firebaugh[9] obtaining instant thickener powder through derivatization pro-
cess at acidic pH. The benefit of obtaining instant thickener powder is to avail the use of
the thickener powder in applications where heating is not required or recommended.
Economical considerations are in favor of starch-based thickeners as compared
with whey protein-based thickeners. The average price of whey protein concentrate in
August 2011 was around USD 3000/ton,[16] while the corn starch one was around USD
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300/ton.[17] The price of whey protein isolate is even higher. The cost of the derivatized
whey protein thickener is expected to be higher than that of native whey proteins as a result
of the processes of thermal treatment, gelation, crushing and freeze drying, or spray drying.
This additional cost will make the product cost prohibitive as compared with starch-based
thickeners.
To avoid the additional cost of gel formation and subsequent drying or derivatiza-
tion, consumers might consider obtaining thickening properties of whey proteins via simple
heating of native proteins to induce in situ thickening in food products during cooking and
serving. For this purpose, in this work, we propose the potential use of whey proteins as a
thickener in food products by simple thermal treatment, which can take place during prod-
uct preparation or cooking. Potential food products include soups, hot drinks, and baked
confectionaries.
The originality of this work does not lie in an innovative preparation methodology,
but in the extensive examination of a wide range of preparation conditions (pH and salt con-
centration), as well as in the introduction of the concept of viscosity contour maps of whey
proteins, in addition to the molecular analysis and characterization of the thickened solu-
tions. This work presents a methodology for controlling the required degree of thickening
by manipulating pH and salt concentration.

MATERIALS AND METHODS

Materials
Commercial whey protein isolate (WPI) (BiPRO) prepared by an ion exchange pro-
cess was kindly supplied from Davisco Food International (LeSueur, MN, USA) and used
as received. Powder composition was 94% protein, 5% moisture, and 1% ash, all by weight.
Distilled water was used in all the experiments.

Preparation of Protein Solutions

Protein solutions were prepared by dissolving the native protein powder in distilled
water by gentle magnetic stirring for 30 min. For each concentration, the pH was adjusted
to two values—pH 3 and pH 7—using 0.1 N HCl and 0.1 N NaOH, respectively. Thermal
treatment of the protein solutions was carried out by heating at 80◦ C for 1 h to denature the
protein and to induce chemical and/or physical bonds, then cooling to 6◦ C and maintaining
at 6◦ C for 24 h.
 572 EISSA ET AL.

Determination of Gel Point

Gel point was determined using the “tilted test tube method,” where the gel point is
determined by tilting a test tube containing the solution. In this approach, the gel point is
determined as the point where the system stops to flow.

Rotational Viscometry
Steady-shear rotational rheological measurements were conducted on a Canon
LV2000 viscometer. In these experiments, samples were placed in temperature-controlled
13-mm diameter test tubes. Spindle L4 was used. The rotational speed ranged from 0.6 to
30 rpm, and viscosity was measured at 30◦ C.
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Intrinsic Viscometry
Cannon Fenske glass capillary viscometers of 50, 100, and 200 sizes were used for
intrinsic viscosity measurements. A cooling-water bath was used to control the temperature
at 30◦ C.

Sodium Dodecyl Sulphonate Polyacrylamide Gel Electrophoresis

(SDS-PAGE)
Polyacrylamide gel electrophoresis (PAGE) was performed using a Mini-PROTEAN
3 Electrophoresis Cell (BIO-RAD, Hercules, CA, USA). Protein solutions were mixed with
40 volumes of sample buffer (40% glycerol, 8% SDS, and the balance being deionized
water). Beta-mercaptoethanol was added (50 μL per 950 μL of sample buffer) when reduc-
ing conditions were needed. The mixture was heated for 5 min at 95◦ C and then subjected to
electrophoresis in 5–15% polyacrylamide gels using the discontinuous system. These gels
were then stained with 0.1% Coomassie blue and then destained by deionized water for 2 h.
The gels were subsequently immersed in drying solution (containing 20% ethanol and 10%
glycerol in deionized water) for 30 min and fixed in drying frames for 24 h. The dry gels
were finally scanned and the bands analyzed in terms of molecular weights. ImageJ soft-
ware (v. 1.44, National Institute of Health, USA) was used in gel analysis. Chromatograms
were then constructed using SigmaPlot software (Systat Software Inc.)

RESULTS AND DISCUSSION

Methodology for Thickener Formation
Since our primary target in this work was to study the thickening properties of whey
protein solutions, we picked two pH values that are far enough from the isoelectric point
to avoid collapse of protein molecules into particulate aggregates.[18] The isoelectric point
of β-LG, the main constituent of whey proteins, is ∼5.1.[19] Hence, pH values were chosen
to be 3 and 7 to prevent excessive collapse of protein molecules. Previous investigations
proved that whey proteins form large aggregates upon thermal treatment at acidic and
neutral pH values.[20,21] Consequently, whey protein solutions at pH 3 and pH 7, at var-
ious salt concentrations, were heated at 80◦ C to induce aggregation and/or polymerization.
Aggregation usually refers to molecular size enlargement via physical interactions while
polymerization refers to molecular size enlargement via chemical reactions.
 CHARACTERIZATION OF WHEY PROTEIN THICKENERS 573

Gel Point at pH 7 and pH 3 at Different Salt Concentrations

In order to determine the suitable range of protein and salt concentrations for thick-
ening, we determined the gel point to avoid gelation. Figure 1 shows the gel points upon
thermal treatment of protein solutions. The thermal treatment involved heating at 80◦ C
for 1 h then cooling to 6◦ C and maintaining at 6◦ C for 24 h. Gelation occurs when pro-
tein molecules polymerize and/or aggregate to form three dimensional networks. Protein
structural changes, polymerization, and aggregation have been discussed extensively in the
literature.[12,18,22–24] Polymerization and aggregation of whey proteins involve two steps:
(1) denaturation, which involves partial—or complete—unfolding of proteins; and (2) asso-
ciation of molecules to form large aggregates.[25] As shown in Fig. 1, at constant salt
concentration, gelation at pH 7 occurred at concentrations lower than at pH 3. At pH 3, ther-
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mal treatment induces no disulphide bonds between β-LG molecules,[26] causing gelation
to occur only by physical interactions. However, the presence of disulphide bonds at pH
7 induces gelation at lower protein concentrations due to the presence of both physical inter-
actions and chemical bonds. In addition, it has been observed that below a certain protein
concentration at pH 3, thermal treatment does not lead to gelation. Instead, it leads to phase
separation of the solution into large aggregate flocs and clear solution. Phase separation
was not observed at pH 7 at any concentration. Phase separation at pH 3 at lower protein
concentrations is believed to stem from the absence of chemical bonds, which appears to
be essential in constructing the gel network at low protein concentrations.

Rotational Viscosity of Heated Whey Protein Solutions

Viscosity was measured at different speeds. Shear rate was evaluated using
Newtonian approximation, which is frequently used in commercial viscometers to

Figure 1 Gel point of heated whey protein solutions (80◦ C for 1 h) at different NaCl concentration at pH
3 and pH 7.
 574 EISSA ET AL.

approximate the shear rate in the form of average shear rate.[27] Since almost all thickeners
are pseudoplastic (shear thinning) fluids, it is important to choose the suitable shear rate
to evaluate the thickening properties. Shear rate in the mouth ranges from 0.1 to 1000 s−1
depending on the viscosity of the food.[28] The higher the viscosity, the lower the shear rate
associated with oral evaluation of viscosity.[28] At viscosities from 1000 to 10,000 cP, the
bound shear rate for oral evaluation ranges from ∼1 to ∼10 s−1 .
Contour maps of viscosity at pH 7 and pH 3 were constructed representing and pre-
dicting the values of viscosity of thermally treated whey protein solutions as a function of
protein concentration and salt concentration, as shown in Figs. 2 and 3. Contour maps were
constructed using Sigma Plot software (version 11). We can deduce from Figs. 2 and 3
that the values of viscosity of thermally treated dispersions at pH 7 are considerably higher
than pH 3 ones. The higher viscosity values at pH 7 can be attributed to the presence of
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disulphide bonds at pH 7, while at pH 3, disulphide bonds are absent and the thickening
properties are solely due to physical interactions.
Figures 4 and 5 show the effect of NaCl concentration on viscosity at different pro-
tein concentrations. At pH 7, the viscosity increases continuously with NaCl concentration
over the whole protein concentration range. However, at pH 3, below a certain protein con-
centration, the viscosity of protein solutions exhibits a maximum. This phenomenon can
be explained in the light of phase separation that occurs at pH 3 below certain protein

Figure 2 Contour lines showing points of same viscosities (cP) of heated whey protein solutions as function of
protein and NaCl concentrations at pH 7. Viscosity is measured at 1 s−1 and 30◦ C.
 CHARACTERIZATION OF WHEY PROTEIN THICKENERS 575
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Figure 3 Contour lines showing points of same viscosities (cP) of heated whey protein solutions as function of
protein and NaCl concentrations at pH 3. Viscosity is measured at 1 s−1 and 30◦ C.

concentration (∼4% by wt.), as discussed above. The non-gelling behavior accompanied

with phase separation at pH 3 below ∼4% causes a decrease in the viscosity, after pass-
ing through a maximum value of viscosity as the solutions separate into clear liquid and
precipitated protein flocs.

Comparison with Food Thickeners

Viscosity values obtained in this work were compared with the viscosity values of
common thickeners reported in the literature. Table 1 shows typical viscosity values of
food thickeners. The values in Table 1 suggest that the obtained viscosity range of whey
protein solutions is very similar to that of the widely used food thickeners in literature.
The viscosity spectrum was also compared with the widely known Milkshake® products
served by McDonalds® restaurants as well as the widely used Egyptian traditional drink of
Orchidaceae. Figure 6 clearly shows that the viscosity patterns of the whey solutions are
very similar to the Milkshake® and Orchidaceae drinks. This behavior suggests that the
whey protein solutions simulate the texture of thickened drinks. Figure 6 also shows that
the viscosity at very low shear rate reaches higher values, approaching 100,000 cP, which
suggests a strong shear thinning behavior. The viscosity values at pH 7 were higher than
at pH 3.
 576 EISSA ET AL.
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Figure 4 Viscosity (cP) of heated whey protein solutions at pH 7 at different NaCl concentrations. Viscosity is
measured at 1 s−1 at 30◦ C.

Figure 5 Viscosity (cP) of heated whey protein solutions at pH 3 at different NaCl concentrations. Viscosity is
measured at 1 s−1 at 30◦ C.
 CHARACTERIZATION OF WHEY PROTEIN THICKENERS 577

Table 1 Typical values of viscosities for food thickeners.

Shear rate, s−1

Thickener Viscosity (cP) unless mentioned Citation

Powdered instant food–thickened 50 rpm (spindle [40]

beverages in: speed)
• Apple juice 2163
• Cranberry 2226
• 2% reduced fat milk 2105
• Orange juice 2473
• Water 2100
Six commercially available food 100–100,000 depending on 1 [41]
thickeners (GuarcolTM , KeltrolTM , thickener type, solvent,
NovartisTM , NutriciaTM ,
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and concentration
QuikThikTM , and SupercolTM )
that are based on guar gum,
modified starch, and xanthan gum
1% carragenan solution 300 9.88 [42]
18.2% reconstituted sweet potatoes 300 1.5 [43]
powder
Thermally treated whey protein 50–3,000 depending on 1 [44]
isolates at pH 3.35 protein concentration

Figure 6 Viscosity of 5% whey protein solutions at pH 7, 60 mM NaCl and pH 3, 120 mM NaCl as compared
with McDonalds® Milkshake® and Orchidaceae drinks. Viscosity is measured at 30◦ C.

Role of Hydrogen Bonding

Hydrogen bonds play an important role in hydrocolloids properties in solutions.[29]
Although they are much weaker than covalent bonds, the population of hydrogen bonds,
especially in polar solvents, may affect the protein solution viscosity to a considerable
 578 EISSA ET AL.
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Figure 7 Effect of urea on viscosity of heated whey protein solutions at pH 7 at 30◦ C. Solid lines show the
viscosity of solutions with no urea addition, while dotted lines show the viscosity after urea addition.

Figure 8 Effect of urea on viscosity of heated whey protein solutions at pH 3 and 30◦ C. Solid lines show the
viscosity of solution before with no urea addition, while dotted lines show the viscosity after urea addition.

extent. To assess the role of hydrogen bonds in the thermally-induced protein polymers, we
used urea to dissociate the hydrogen bonds. Urea is known as a hydrogen-bond-breaking
reagent. Figures 7 and 8 show the effect of urea on solution viscosity at pH 7 and pH 3,
respectively. Figure 7 shows that hydrogen bonds play a minor role in the thickening effect
 CHARACTERIZATION OF WHEY PROTEIN THICKENERS 579

of polymerized whey protein solutions at pH 7. However, at pH 3 (Fig. 8), viscosity drops

dramatically upon addition of urea. This confirms that hydrogen bonding in whey protein
solutions at pH 3 is a major mechanism for the thickening properties, which agrees with
the fact that at pH 3, no disulphide bonds are created upon thermal treatment.

Intrinsic Viscosity
Dilute solution viscometry is another valuable tool that can be used to probe the
molecular properties of the thickening agent.[14] One of the most salient features of macro-
molecules is the intense increment in viscosity that they produce when, even in minute
amounts, they are dissolved in ordinary solvents. The viscosity intensifying effect, charac-
terized by the intrinsic viscosity, [η], is extensively used for analysis or characterization of
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synthetic polymers,[30] biological macromolecules, nanoparticles, and colloids.

Intrinsic viscosity [η], provides information about fundamental properties of the
solute and its interaction with the solvent. It can also be precisely related to the confor-
mation of flexible (linear and non-linear) chains, wormlike macromolecules and micelles,
and rigid particles of arbitrary shape. The intrinsic viscosity is defined as:

ηsp ηr − 1
[η] = lim = lim , (1)
c→0 c c→0 c

where [η], ηsp , ηr , and c are the intrinsic viscosity, specific viscosity, relative viscosity and
concentration, respectively. A classical procedure for its determination, based on Eq. (1),
consists of the determination of viscosity of solutions at various concentrations, followed
η
by extrapolation of csp to zero concentration. In a range of moderate concentrations, the
dependence is linear and can be written as the Huggins equation:

ηsp
= [η] + kH [η]2 c, (2)
c

where kH is the (dimensionless) Huggins constant. Thus, the intrinsic viscosity [η] can
be obtained as the intercept in a linear least-squares fit. Intrinsic viscosity is measured for
very dilute solutions, and thus it gives an idea of the polymer’s molecular size and shape per
unit volume, excluding the effect of hydrogen bonding, which is weakened due to excessive
dilution.
The intrinsic viscosity of whey protein polymers in the current work reached up to
∼0.6 dl/g (Figs. 9 and 10, Table 2). Values of ∼0.05 dl/g were reported for native whey
proteins.[31] Thus, polymerized whey proteins show an ∼10-fold increase in the intrinsic
viscosity as compared with the native whey proteins. The values of the intrinsic viscos-
ity are in accordance with previously-published intrinsic viscosity data for whey protein
polymers.[32]
According to Imeson,[33] Guar solutions have an intrinsic viscosity range of
0.7–15 dl/g. Montana polysaccharides[34] declared Levan to be one of the most efficient
thickeners with intrinsic viscosity of 0.14 dl/g. Another type of thickener, Carboxymethyl
cellulose, when dissolved in water, exhibits values of intrinsic viscosity of 0.32 dl/g.[35]
Table 3 shows the values of intrinsic viscosity for different food thickeners. Such values of
intrinsic viscosity are close to the values obtained in this work, and thus suggest that whey
protein can be a viable food thickener.
 580 EISSA ET AL.
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Conc (g/dl)
Figure 9 Intrinsic viscosity (dl/g) of heated whey protein solutions at pH 3 at different NaCl concentrations.
Viscosity is measured at 30◦ C.

Conc (g/dl)
Figure 10 Intrinsic viscosity (dl/g) of heated whey protein solutions at pH 7 at different NaCl concentrations.
Viscosity is measured at 30◦ C.
 CHARACTERIZATION OF WHEY PROTEIN THICKENERS 581

Table 2 Intrinsic viscosity and kH values of heated whey protein solutions at pH 3 and pH 7 at different salt
concentrations.

pH 7 pH 3

NaCl Conc.
WPI Conc. % (mM) [η] dL/g K WPI Conc. % Salt (mM) [η] dL/g K

7 20 0.48 0.14 7 20 0.61 0.72

6.5 30 0.31 0.46 6 24 0.37 0.41
2 140 0.01 10,300 5.5 50 0.46 4.10

Table 3 Typical intrinsic viscosity values of different thickeners.

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Thickener Intrinsic viscosity (dl/g) Citation

Guar products 0.7–15 [33]

WPC 0.6 [32]
Levan 0.14 [34]
Carboxymethyl cellulose 0.32 [45]

Table 4 Suggested molecular structures of the heated whey protein solutions, on the light of their kH values.

pH WPI Conc. % NaCl Conc. (mM) kH Predicted system type

7 7 20 0.14 Flexible, unbranched chains

7 6.5 30 0.46 Flexible, unbranched rod-like molecules
7 2 140 10,300 Aggregating, interacting macromolecules
3 7 20 0.72 Globular particles
3 6 24 0.41 Flexible, worm-like particles
3 5.5 50 4.1 Aggregating macromolecules

Huggins constant and chain conformation. Huggins constant provides use-

ful information in predicting the conformation of the polymeric systems. An extensive
literature compilation of kH values for a variety of systems was found in the poly-
mer handbook.[36] Flexible polymer chains possess kH values in the range 0.2–0.8; most
frequently about 0.3 in good solvents.[37] For rod-like particles, experimental data for well-
characterized, non-associating rod-like macromolecules and particles yield values in the
range of kH ≈ 0.4–0.7. Since this range is similar to the range expected from fully flexible
chains, one expects and the experiment confirms that wormlike chains should have such kH
range from the coil limit to the rod limit.[37]
From results presented in Table 4, we can deduce that the samples of salt concentra-
tion less than ∼30 mM contain protein in flexible-chain conformations when comparing
their kH values with those in the literature.[38] On the other hand, samples of higher salt
concentration exhibit very large values of kH , which indicate the condition for aggregat-
ing molecules.[36] This can be attributed to the high salt content that leads to screening of
electrostatic repulsion, yielding collapsed, aggregated molecules.
Molecular weight profile by electrophoresis. Four samples, in addition to
molecular weight market were analyzed by SDS-PAGE electrophoresis as follows:
r Lane 1: Molecular weight marker;
r Lane 2: 7.5%, 20 mM NaCl, pH 7 (sample 1);
 582 EISSA ET AL.

r Lane 3: 6.5%, 50 mM NaCl, pH 7 (sample 2);

r Lane 4: 8%, 60 mM NaCl, pH 3 (sample 3);
r Lane 5: 7%, 72 mM NaCl, pH 3 (sample 4).

The experiment was undergone twice, once without β-mercaptomethanol (non-reducing

conditions), and the other run was done by adding β-mercaptoethanol (reducing condi-
tions), which dissociates the disulfide bonds.

Analysis of Electrophoresis Gel under

Non-Reducing Conditions
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The addition of SDS breaks the hydrophobic associations due to its surface activity
and ability to attach to both hydrophilic and hydrophobic residues. Figure 11 shows a high
molecular weight shoulder at the most left of the graph at pH 7 (samples 1 and 2), indicative
to the very top of the gel. Such profile indicates a molecular weight ∼ ≥1 million kDa.[18]
However, we see the samples at pH 3 (samples 3 and 4) exhibiting a wide distribution of
molecular weight, noticeably lower than samples at pH 7. The absence of disulfide bonds at
pH 3 has been reported in the literature.[39] However, it is apparent from lanes 4 and 5 that
aggregation still exists despite using SDS. Such aggregation at pH 3 can be explained by
two ways: (1) either that the amount of SDS added was not enough to dissociate the whole
sample and break all its hydrophobic bonds, and thus some high molecular weight pro-
tein were left aggregated; or (2) hydrophobic interactions in the samples formed compact
micelles and secondary structures in the micelle cores. Whereas nonpolar side chains are
driven into the interior of the protein, most polar side chains remain in contact with water
on the surface of the protein. The sections of the polar backbone that are forced into the
interior of the protein neutralize their polarity by hydrogen bonding to each other, often
generating secondary structures. Studies of folding pathways indicate that hydrophobic col-
lapse and formation of secondary structures occur simultaneously.[39] Aggregation of the
hydrophobic chains in the protein interior giving rise to the formation of a compact globule
or a micelle, could have happened upon heat treatment of the samples in the first place,
and remained stable throughout, even after addition of SDS. This means that the protein
micelles interacted with their outer surface keeping the interior partially protected. If such
a theory stands, it surely would affect the overall protein dissociation upon the addition of
SDS, and would cause high molecular weight aggregates.

Analysis of Electrophoresis Gel under Reducing Conditions

Adding β-mercaptoethanol breaks the accessible disulphide bonds in protein sam-
ples. Figure 12 shows SDS-PAGE chromatograms of the four protein samples described
earlier. We clearly see the curves of samples at pH 7 (samples 1 and 2) to be monomers,
dimmers, and oligomers of β-LG and β-lactalbumin, indicative of cleavage of most of disul-
fide bonds that were responsible for the high molecular weight band at the top of the gel
(left most of the curve) under non reducing conditions (Fig. 11). Samples at pH 3 (samples
3 and 4) appear similar to samples 1 and 2, indicating that the cleavage of intramolecular
disulfide bonds has eased the access of SDS to hydrophobic associations in the micelles that
were responsible for high molecular weights shown by chromatograms of samples 3 and
4 in Fig. 11.
 CHARACTERIZATION OF WHEY PROTEIN THICKENERS 583

Top of Bottom of
the Gel the Gel

MW Marker 19 kDa
119 kDa
90 kDa 50 kDa
34 kDa 26 kDa

Sample 1
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Sample 2

Sample 3

Sample 4

Figure 11 Chromatograms of SDS-PAGE for five samples: M.Wt. marker; Sample 1: 7.5%, 20 mM NaCl, pH 7;
Sample 2: 6.5%, 50 mM NaCl, pH 7; Sample 3: 8%, 60 mM NaCl, pH 3; Sample 4: 7%, 72 mM NaCl, pH 3.
(Color figure available online.)

CONCLUSIONS
A simple methodology of heat treatment of whey protein isolate solutions led to the
formation of high molecular polymers capable of introducing thickening properties in food
or pharmaceutical products. The presence of chemical (disulphide) bonds in heated protein
solutions at pH 7 resulted in higher viscosity as compared with pH 3, where disulphide
bonds are absent. Viscosity contours at pH 3 and pH 7 were constructed to show the vis-
cosity at different protein and salt concentrations. The higher the protein concentration, the
lower the salt concentration needed to reach the required thickening properties. Phase sep-
aration occurred at lower protein concentrations at pH 3, presumably due to the absence
of disulphide bonds. Comparison with some available thickened food products was held
to show that protein solutions have similar values of the viscosity. The work demonstrated
potential use of whey proteins as a thickener in a wide variety of food products. The intrinsic
 584 EISSA ET AL.

Top of Bottom of
the Gel the Gel

MW Marker 19 kDa
119 kDa 90 kDa 50 kDa
34 kDa 26 kDa

Sample 1
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Sample 2

Sample 3

Sample 4

Figure 12 Chromatograms of SDS-PAGE under reducing conditions (with β-mercaptoethanol) for five samples:
M.Wt. marker; Sample 1: 7.5%, 20 mM NaCl, pH 7; Sample 2: 6.5%, 50 mM NaCl, pH 7; Sample 3: 8%, 60 mM
NaCl, pH 3; Sample 4: 7%, 72 mM NaCl, pH 3. (Color figure available online.)

viscosity measurements showed that the whey protein polymers tend to exhibit a flexi-
ble chain conformation at lower ionic strength; while at high ionic strength, the protein
molecules exhibit a collapsed and aggregated structure. By comparing the kH values with
that of other substances, they fell very near to the range of the polysaccharide-based thick-
eners. Electrophoresis results indicated a possible micelle configuration for heated whey
proteins at pH 3, possibly aided by intramolecular disulphide bonds.

REFERENCES
1. Regster, G.O.; Mcintosh, G.H.; Lee, V.W.K.; Smithers, G.W. Whey proteins as nutritional and
functional ingredients. Food Australia 1996, 48, 123–127.
2. Bounous, G.; Gervais, F.; Amer, V.; Batist, G.; Gold, P. The influence of dietary whey protein
on tissue glutathione and the diseases of aging. Clinical and Investigative Medicine 1989, 12,
343–349.
 CHARACTERIZATION OF WHEY PROTEIN THICKENERS 585

3. Kennedy, R.S.; Konok, G.P.; Bounous, G.; Barouchel, S.; Lee, T.D. The use of whey protein
concentrate in the treatment of patients with metastatic carcinoma. Anticancer Research 1995,
15, 2643–2649.
4. Fox, P.F. The milk protein system. In: Developments in Dairy Chemistry, Vol. 4; London:
Elsevier Science Publishers, 1989; 1–53.
5. McIntosh, G.H.; Regester, G.D.; Lelue, R.K.; Royle, P.Y.; Smithers, G.W. Dairy proteins protect
against dimethylhydrazine-induced intestinal cancers in rats. Journal of Nutrition 1995, 125,
809–816.
6. Jost, R. Functional characteristics of dairy proteins. Trends in Food Science Technology 1995, 4,
283–288.
7. Monahan, F.J.; German, J.B.; Kinsella, J.E. Effect of pH and temperature on protein unfolding
and thiol/disulfide interchange reactions during heat-induced gelation of whey proteins. Journal
Downloaded by [University of Nebraska, Lincoln] at 13:56 14 October 2014

of Agricultural and Food Chemistry 1995, 43, 46–52.

8. Mleko, S.; Foegeding, E.A. Formation of whey protein polymers: Effects of a two-step heating
process on rheological properties. Journal of Texture Studies 1999, 30, 137–149.
9. Firebaugh, J.D. Characterization and application of a derivatized whey ingredient. Ph.D. Thesis,
North Carolina State University, Raleigh, NC, 2004.
10. Holst, H.H.; Albertsen, K.; Clausen, P.M.; Thomsen, B.; Hartmann, U. Partly denatured whey
protein product. PCT/EP93/1093, 1993.
11. Thomsen, B. Whey protein texturizer. European Food and Drink Review 1994, 46–47.
12. Elofsson, C.; Dejmek, P.; Paulsson, M.; Burling, H. Characterization of a cold-gelling whey
protein concentrate. International Journal of Dairy Science 1998, 7, 601–608.
13. Hudson, H.M.; Daubert, C.R.; Foegeding, E.A. Rheological and physical properties of derivitized
whey protein isolate powders. Jouranl of Agricultural and Food Chemistry 2000, 48, 3112–3119.
14. Resch, J.J.; Daubert, C.R. Rheological and physicochemical properties of derivatized whey
protein concentrate powder. International Jouranl of Food Properties 2002, 5 (2), 419–434.
15. Resch, J.J.; Daubert, C.R.; Foegeding, E.A. A comparison of drying operations on the rheological
properties of whey protein thickening ingredients. International Journal of Food Science and
Technology 2004, 39, 1023–1031.
16. Gould, B.W. Dry milk product price. Available online at: http://future.aae.wisc.edu/data/
weekly_values/by_area/1610?tab=prices (accessed 27 August 2011).
17. Barrientos, M. Maize (corn) daily price. Available online at: http://www.indexmundi.com/
commodities/?commodity=corn (accessed 27 August 2011).
18. Eissa, A.S. Enzymatic modification of whey protein gels in low pH. Ph.D. Thesis, North Carolina
State University, Raleigh, NC, 2005.
19. Verheul, M. Aggregation and gelation of whey proteins. Ph.D. Thesis, Universiteit Twente,
Enschede, The Netherlands, 1997.
20. Bolder, S.G.; Hendrickx, H.; Sagis, L.M.; van der Linden, E. Fibril assemblies in aqueous whey
protein mixtures. Journal of Agricultural and Food Chemistry 2006, 54, 4229–4234.
21. Suzanne, G.B.; Astrid, J.V.; Leonard, M.C.; Erik, V.L. Heat-induced whey protein isolate fibrils:
Conversion, hydrolysis, and disulphide bond formation. International Dairy Journal 2007, 17,
846–853.
22. Griffin, W.G.; Griffin, M.C.; Martin, S.R. The molecular basis of thermal aggregation of bovine
beta-lactoglobulin A. Journal of Chemistry Society Faraday Transactions 1993, 89, 3395–3406.
23. Elofsson, U.M.; Dejmek, P.; Paulsson, M.A. Heat-induced aggregation of β-lactoglobulin studied
by dynamic light scattering. International Dairy Journal 1996, 6, 343–357.
24. Iametti, S.; De Gregori, B.; Vecchio, G.; Bonomi, F. Modifications occur at different structural
levels during the heat denaturation of β-lactoglobulin. European Journal of Biochemistry 1996,
237 (1), 106–112.
25. Mulvihill, D.M.; Donovan, M. Whey proteins and their thermal denaturation—A review. Irish
Journal of Food Science and Technology 1987, 11, 43–75.
 586 EISSA ET AL.

26. Errington, A.D.; Foegeding, E.A. Factors determining fracture stress and strain of fine-stranded
whey protein gels. Journal of Agricultural and Food Chemistry 1998, 46, 2963–2967.
27. Steffe, J.F. Rheological Methods in Food Process Engineering; Freeman Press: East Lansing,
MI, 1996.
28. Sharma, F.; Sherman, P. Identification stimuli controlling the sensory evaluation of viscosity II:
Oral methods. Journal of Texture Studies 1973, 4, 111–118.
29. Ross-Murphy, S.B. Rheological characterisation of gels. Journal of Texture Studies 1995, 26,
391–400.
30. Elias, H.G. Macromolecules; New York: Plenum Press, 1977.
31. Vardhanabhuti, B.; Foegeding, E.A. Rheological properties and characterization of polymerized
whey protein isolates. Journal of Agricultural and Food Chemistry 1999, 49 (9), 3649–3655.
32. Resch, J.J.; Daubert, C.R.; Hudson, H.M. Rheological Characterization of Derivatized Whey
Downloaded by [University of Nebraska, Lincoln] at 13:56 14 October 2014

Protein Concentrate Solutions; NC State University: Raleigh, NC, 2002.

33. Imeson, A. Food Stabilisers, Thickeners and Gelling Agents; Wiley Blackwell: Oxford, 2010.
34. Montana Polysaccharides Corp. About Levan: Rheology. Available online at: http://www.
polysaccharides.us/ (accessed 27 August 2011).
35. Sagar, P.; Mal, D.; Singh, R.P. Synthesis and characterization of cationic guar gum: A high
performance flocculating agent. Journal of Applied Polymer Science 2007, 105, 3240–3245.
36. Billmeyer, F. Textbook of Polymer Science; Wiley Interscience: NewYork, 1984.
37. Ramon Pamies, J.H.M.G. Determination of intrinsic viscosities of macromolecules and nanopar-
ticles. Comparison of single-point and Dilution procedures. Colloid Polymer Science 2008, 286,
1223–1231.
38. Kurata, M.; Tsunashima, Y. Viscosity-Molecular Weight Relationships and Unperturbed
Dimensions of Linear Chain Molecules; Wiley: New York, 1989.
39. Horton, M. Principles of Biochemistry; Saddle River, NJ: Prentice Hall, 2002.
40. Adeleye, B.; Rachal, C. Comparison of the rheological properties of ready-to-serve and powdered
instant food–thickened beverages at different temperatures for dysphagic patients. Journal of the
American Dietetic Association 2007, 107 (7), 1176–1182.
41. Sopade, P.A.; Halley, P.J.; Cichero, J.A.Y.; Ward, L.C. Rheological characterisation of food
thickeners marketed in Australia in various media for the management of dysphagia. I: Water
and cordial. Journal of Food Engineering 2007, 79, 69–82.
42. Prentice, J.H.; Huber, D. Results of collaborative study on measuring rheological properties
of foodstuffs. In: Physical Properties of Foods; London: Applied Science Publishers, 1983;
123–184.
43. Grabowskia, J.A.; Truong, V.D.; Daubertb, C.R. Nutritional and rheological characterization of
spray dried sweet potato powder. LWT–Food Science and Technology 2008, 41, 206–216.
44. Daubert, C.R.; Hudson, H.M.; Foegeding, E.A.; Prabhasankar, P. Rheological characterization
and electrokinetic phenomena of charged whey protein dispersions of defined sizes. LWT–Food
Science and Technology 2006, 39, 206–215.
45. Sagar Pal, D.M.R.P.S. Synthesis and characterization of cationic guar gum: A high performance
flocculating agent. Journal of Applied Polymer Science 2007, 3240–3245.

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