Food Hydrocolloids 63 (2017) 130e138

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Whey proteinepectin soluble complexes for beverage applications

Ty B. Wagoner, E. Allen Foegeding*
Department of Food, Bioprocessing and Nutrition Sciences, Box 7624, North Carolina State University, Raleigh, NC 27695-7624, USA

a r t i c l e i n f o a b s t r a c t

Article history: There is a strong interest in the consumption of beverages containing whey proteins due to implications
Received 2 May 2016 in health outcomes such as increased satiety and metabolic regulation. However, low thermal stability
Received in revised form limits the conditions under which whey protein beverages can be formulated. Studies have shown that at
8 August 2016
a narrow pH range near the protein isoelectric points, whey proteins and polysaccharides self assemble
Accepted 22 August 2016
into soluble complexes (SCs) that exhibit unique functionality. This study investigated the formation and
Available online 24 August 2016
thermal stability of SCs under conditions relevant to beverage applications. Complexes were formed at
pH 5 using whey protein isolate (WPI; 1e6% w/w) and high-methoxyl pectin (HMP; 0.125e0.75% w/w)
Keywords:
Whey protein
and then heat-set at 85
C for 25 min. Hydrodynamic properties, particle size distribution, zepotential,
Polysaccharides and dispersion viscosity were evaluated before and after heat-setting. Mean particle diameter ranged
Rheology from 300 to 715 nm for unheated SCs, and 230 nm to 1 mm for heat-set SCs. Heat-setting SCs led to a
Complexes significant (p < 0.05) reduction in intrinsic viscosity from 93.6 mL/g to 79.5 mL/g, suggesting confor-
Beverages mational changes that favor a smaller hydrodynamic size. Dispersions of SCs exhibited decreased
Thermal stability apparent viscosity, consistent with the lower intrinsic viscosity and smaller particle size. Heat-set SCs (4%
WPI, 0.5% HMP) remained as sub-micron particles (d ¼ 303e829 nm) after pH adjustment (pH 4e7) and
thermal processing (142
C for 6 s), indicating that WPI and HMP can be heat-set into complexes with
enhanced colloidal stability in beverage applications.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction (pI) of whey proteins, where protein precipitation, microphase

There is strong interest in the consumption of whey proteins
due to potential health benefits, including increased satiety,
metabolic regulation, and as part of a weight loss intervention
€rck, 2007; Zafar,
program (Hector et al., 2015; Nilsson, Holst, & Bjo
Waslien, AlRaefaei, Alrashidi, & AlMahmoud, 2013). One partic-
ular category of interest is protein beverages, such as meal-
replacement beverages or recovery sports drinks. Beverages
require thermal processing for safety and shelf stability, but whey
proteins are notoriously fastidious to heat, particularly at pH or
ionic strength where electrostatic stabilization is diminished.
Currently, stable beverages are formulated under acidic or neutral
conditions, where astringency and off-flavors respectively are of
concern (Beecher, Drake, Luck, & Foegeding, 2008; Evans,
Zulewska, Newbold, Drake, & Barbano, 2010). To mitigate senso-
rial defects, whey protein beverages would ideally be formulated at
pH 4e6. However, this pH range encompasses the isoelectric point
* Corresponding author.
E-mail address: allen_foegeding@ncsu.edu (E.A. Foegeding).
separation, or gelation may occur after heating (Ako, Nicolai,
Durand, & Brotons, 2009; Langton & Hermansson, 1992).
One method of influencing whey protein thermal stability is via
the formation of whey protein and polysaccharide complexes
(Turgeon, Schmitt, & Sanchez, 2007). Complexation occurs as a
result of electrostatic interactions between anionic polysaccharides
and positively charged surface patches (notably eNHþ 3 groups) on
the proteins (Cooper, Dubin, Kayitmazer, & Turksen, 2005;
Tolstoguzov, 2003). This “charge patch” mechanism may also be
influenced by charge regulation and ion-dipole interactions at pH
near the pI (da Silva & Jo €nsson, 2009). Depending on the electro-
static conditions, the complexes will remain as small, soluble
complexes (SCs) or undergo associative or segregative phase sepa-
ration (Cooper et al., 2005; de Kruif, Weinbreck & de Vries, 2004;
Tolstoguzov, 2003; Turgeon et al., 2007; Weinbreck, Wientjes,
Nieuwenhuijse, Robijn, & Kruif, 2004).
Soluble complexes have previously been shown to exhibit
functionality in a number of food applications (Champagne &
Fustier, 2007; Liu, Xu, & Guo, 2007; Schmitt & Turgeon, 2011).
This represents a unique opportunity in beverage applications,
where to our knowledge the use of soluble complexes has not been

http://dx.doi.org/10.1016/j.foodhyd.2016.08.027
0268-005X/© 2016 Elsevier Ltd. All rights reserved.
 T.B. Wagoner, E.A. Foegeding / Food Hydrocolloids 63 (2017) 130e138
explored. Previous research indicates that SCs are formed over the
pH range 4e6 (Gente s, St-Gelais, & Turgeon, 2010; Jones &
McClements, 2011; Krzeminski, Prell, Weiss, & Hinrichs, 2014;
Salminen & Weiss, 2013). Their physical characteristics can be
optimized via judicious selection of pH, polymer ratio, ionic
strength, ionic species, polysaccharide charge density, and cosol-
vents (Chanasattru, Jones, Decker, & McClements, 2009; Girard,
Turgeon, & Gauthier, 2002; Hirt & Jones, 2014; Murphy, Cho,
Farkas, & Jones, 2015; Salminen & Weiss, 2013; Sperber, Cohen
Stuart, Schols, Voragen, & Norde, 2009). Moreover, SCs exhibit
stability to a heat-setting step above protein denaturation tem-
peratures and under conditions normally associated with low sta-
bility, such as pH near the pI or high salt (Jones & McClements,
2008; Krzeminski et al., 2014). The heat-setting step directs pro-
tein aggregation and complexation towards the formation of par-
ticles of a discrete size ranging from 225 to 540 nm depending on
formation conditions (Jones & McClements, 2010; Salminen &
Weiss, 2013). However, most of these studies formed complexes
at low protein concentrations e typically 0.5% or below (as reported
in review by Jones & McClements, 2011). In order to meet FDA re-
quirements for “high” or “excellent source of protein” claims,
beverages would need to contain 10 g protein per serving, or ~4%
protein per 250 mL (Code of Federal Regulations, 2016 title 21, sec
101.54).
To be commercially viable, SCs need to be prepared with com-
mon food ingredients and economically feasible processing oper-
ations. In this study, whey protein isolate (WPI) and high-methoxyl
pectin (HMP) were selected due to current widespread usage in
beverages. Whey protein isolate is a value-added ingredient con-
taining 90% protein, with the mixture and amount of individual
proteins dependent on the fractionation technique (Huffman &
Harper, 1999). Whey protein isolate derived from sweet whey is
comprised of ~50% belg, ~15% aelactalbumin, ~20% glyco-
macropeptide (GMP), and small amounts of bovine serum albumin,
lactoferrin, and immunoglobulins (Farrell et al., 2004). Pectin is an
anionic polysaccharide derived from acid extraction of plant ma-
terials, most commonly apple pulp or citrus peel. All pectins consist
of a linear backbone of repeating Degalacturonic acid monomers
linked by ae1e4 glycosidic bonds. The presence of neutral sugar
side chains and branching are also present to some extent and vary
by pectin source. The pKa of carboxyl moieties ranges from 2.9 to
3.3 depending on pectin source, and ~70% are naturally methylated
(Whistler & BeMiller, 1993). Pectin with 50% methylation is
classified as HMP. Pectin is commonly used to stabilize acidic
casein-based dairy beverages where it adsorbs to the surface of
casein micelles, providing steric and electrostatic stabilization
(Lopes da Silva & Rao, 2006; Pereyra, Schmidt, & Wicker, 1997;
Tromp, de Kruif, van Eijk, & Rolin, 2004).
The primary requirements for beverage applications are high
protein concentration, stability to thermal processing, appropriate
viscosity and sensorial attributes, and post-processing shelf sta-
bility. Therefore, the specific goals of this study were to determine
the WPI and HMP concentrations over which SCs could be formed
and evaluate properties relevant to their use in beverages,
including size distribution, hydrodynamic properties, surface
charge, and flow behavior. Additionally, the use of heat-set com-
plexes in a model beverage was explored.
2. Materials and methods
2.1. Materials
Whey protein isolate (WPI) was provided by Glanbia Nutri-
tionals (Twin Falls, ID, USA). Protein content was 92.13% as deter-
mined by Kjeldahl analysis (N  6.38). The amount of individual
131
whey proteins as determined by the manufacturer was 51.2% belg,
21.3% aelactalbumin, 24.3% GMP, 1.6% immunoglobulins, 0.8%
bovine serum albumin and 0.2% lactoferrin. Mineral composition,
as determined by inductively coupled plasma atomic emission
spectroscopy, was 14.4% N, 0.3% P, 0.4% K, 0.6% Ca, 1.1% S, and 0.2%
Na. High-methoxyl pectin (71.8% esterified as reported by the
manufacturer, <1% ash) was provided by CP Kelco (Atlanta, GA,
USA). Chemical reagents were of analytical grade and supplied by
Sigma-Aldrich (St. Louis, MO, USA). All reported protein concen-
trations account for ingredient purity and are expressed as con-
centration of protein, not concentration of WPI powder.
2.2. Preparation of solutions and formation of soluble complexes
Stock protein solutions (10% w/w protein) were formed by sol-
ubilizing WPI in deionized water (>17 MU) by stirring at 300 rpm
for 3 h at ambient temperature (23 ± 1
C), then stored at 5
C
overnight. Stock HMP solutions (2% w/w) were formed by stirring
HMP in deionized water at 600 rpm, heating to 70
C to hydrate,
and stirring at 400 rpm for 6 h at ambient temperature (23 ± 1
C).
Stock solutions were diluted to final concentrations with deionized
water and adjusted to appropriate pH using 1 M citric acid or 1 M
NaOH. Immediately prior to the formation of complexes, pectin and
WPI solutions were adjusted to pH 7. The SCs were formed at an 8:1
mass ratio of protein to pectin. This ratio was identified in a pre-
liminary study (data not shown) as forming the smallest particles,
and agrees with the results of Jones, Decker, and McClements
(2009).
The SCs were formed by first combining the pH 7 stock solutions
at an 8:1 mass ratio of protein to pectin and diluting to the desired
concentration with deionized water. The pH was then adjusted to 5
with 1 M citric acid to form SCs. Heat-set SCs were formed by
heating solutions in an 85
C water bath for 25 min. After heating,
solutions were cooled in an ice slurry and stored overnight at 5
C
before analysis.
2.3. Intrinsic viscosity
Intrinsic viscosity of polymer solutions was measured using a
Cannon-Fenske (Cannon Instruments, State College, PA) capillary
viscometer per the method described by Vardhanabhuti and
Foegeding (1999) with several modifications. Viscometers were
immersed in a temperature controlled water bath set at 40 ± 0.2
C.
Deionized water was used to dilute the stock protein solution to
five concentrations between 0.01 and 0.16% w/w. The pH was
adjusted, if needed, after dilution of the SCs; however, the ionic
strength was not adjusted in order to keep the protein:salt ratio
constant. Pectin was prepared and diluted in 50 mM NaCl to five
concentrations between 0.0125 and 0.2% w/w. For all measure-
ments, 7 mL of sample were added to the viscometer and allowed to
equilibrate for 10 min. Two measurements were performed 10 min
apart and averaged for each data point. Viscometers were rinsed
three times with deionized water and once with acetone before
being completely dried between runs. Specific viscosity (hsp) and
relative viscosity (hrel) were calculated using Eqs. (1) and (2),
respectively, where t is the efflux time of the sample solution and t0
is the efflux time of the solvent (Kragh, 1961).
t  t0
hsp ¼ (1)
t0
t
hrel ¼ (2)
t0
Intrinsic viscosity [h] was determined by plotting hsp/c and hrel/
132 T.B. Wagoner, E.A. Foegeding / Food Hydrocolloids 63 (2017) 130e138
c as a function of polymer concentration via extrapolation of the
best-fit line to zero polymer concentration on the basis of the
Huggins (Eq. (3)) and Kraemer (Eq. (4)) equations, where kH is the
Huggins constant for a given solvent, kK is the Kramer constant for a
given solvent, c is polymer concentration, and [h] is intrinsic vis-
cosity (Tanford, 1961).
hsp
¼ ½h þ kH ½h2 c
c Viscosity flow profiles were developed via shear rate sweeps at
lnðhrel Þ
¼ ½hc þ kK ½h2 c
c mode. A double gap concentric cylinder attachment was filled with
This technique is valid for solutions with hrel values up to ~ 2. All hrel
values were less than 2 with the exception of 0.16% SCs, which had
hrel values less than 2.8. These values fit the model per the Huggins
and Kramer equations and were included in subsequent
calculations.
2.4. Laser diffraction particle size analysis
The size distribution of SCs was determined using laser
diffraction on a Mastersizer 3000 with Hydro EV sample interface
(Malvern Instruments Ltd., Worcestershire, UK). Size calculations
were made using the Mie scattering model for spherical particles.
Water was used as a dispersion solvent with a refractive index of
1.333, and a refractive index of 1.541 and absorbance of 0.001 was
used for SCs. Sample solutions were added to reach an obscuration
level of 10 ± 1% and stirred at 2000 rpm for the duration of the test.
The instrument measured the volume fraction in each of 100
separate size classes ranging from 0.01 to 3000 mm. Particle size
results are expressed as 50th and 90th percentile values (D50 and
D90) and volume mean diameter D[4,3] (Eq. (5)), where ni is the
number of particles of diameter di. Reporting various size classes
allowed for statistical analysis among treatments.
P an ice slurry for 1 min and allowed to equilibrate overnight at 5
C
ni d4i
D½4;3 ¼ P
ni d3i
2.5. Dynamic light scattering (DLS) particle size analysis and
zepotential
Intensity-weighted z-average diameter and zepotential were
measured at ambient temperature (23 ± 1
C) simultaneously using
a Wyatt Mo €biuz (Wyatt Technology, Santa Barbara, CA). Samples
were diluted to a protein concentration of 1 mg/mL to reduce
multiple scattering effects. The pH was adjusted, if needed, after
dilution. To calculate particle diameter using DLS, fluctuations due
to Brownian motion were measured over 30 s intervals and quan-
tified per a second order correlation function with a regularization
fit in the accompanying software (Dynamics v7.3.1, Wyatt Tech-
nology). The calculated diffusion coefficient is related to the hy-
drodynamic diameter per the Stokes-Einstein equation. Mass
concentration (cmass) was used to approximate the mass percentage
of particles in distinct size ranges based on the measured scattering
intensity (Eq. (6)), where I is the intensity of scattered light, M is
molar mass, P is a form factor, r is particle radius, and q is the
angular dependence of scattered light.
Iðr; qÞ
cmass f
MðrÞ  Pðr; qÞ
The Rayleigh-Gans approximation for Rayleigh spheres was
used as a form factor, which assumes molar mass is proportional to
the cube of particle radius. zePotential was calculated using phase
analysis light scattering. A voltage of 3 V was applied at 10 Hz, and
five measurements were averaged over 20 s periods in order to
calculate electrophoretic mobility. Electrophoretic mobility is
related to zepotential by the Smoluchowski equation.
2.6. Apparent viscosity
(3)
ambient temperature (23 ± 1
C) using a controlled stress rheom-
(4) eter (Anton Paar MCR 302, Graz, Austria) under controlled rate
3.8 mL sample and pre-sheared for 15 s at 10 s1. The sample was
held at zero shear for 10 s before a linear shear rate ramp was
applied from 0.1 to 200 s1. Flow profiles were modeled using the
Power Law model (Eq. (7)) for fluids where K represents the flow
consistency coefficient (Pa sn), n represents the dimensionless flow
behavior index, h represents apparent viscosity (Pa s), and g_ rep-
resents shear rate (s1).
h ¼ K g_ n1 (7)
2.7. Ultra high temperature (UHT) thermal processing
Soluble complexes were prepared and heat-set at pH 5 as
described above. The pH was then adjusted to 3, 4, 6 or 7 using 1 M
citric acid or 1 NaOH. Solutions were heat-treated per the method
previously described by Wagoner, Ward, and Foegeding (2015).
Briefly, 2 mL samples were equilibrated to 23
C in capped boro-
silicate test tubes and processed in a 150
C oil bath under manual
agitation. Solution temperature reached 141
C in 62 s and was then
held at 141
C for 6 s to mimic thermal processing commonly used
for protein beverages. After the hold time, test tubes were cooled in
before analysis.
(5)
2.8. Experimental design
All data are from three independent experimental replications
unless stated otherwise. Where applicable, mean values are
given ± standard deviation. Statistical analysis was performed us-
ing the analysis of variance procedure in JMP v12.0 (SAS Institute,
Cary, NC, USA). Statistical significance was indicated at p < 0.05.
Differences among means were determined using Tukey's HSD post
hoc comparison test.
3. Results and discussion
3.1. Intrinsic viscosity
Intrinsic viscosity, [h], represents the contribution to viscosity
by a dispersed polymer and is proportional to hydrodynamic vol-
ume (Lopes da Silva & Rao, 2006). The [h] of HMP and WPI were
measured independently for comparative purposes, and for SCs to
evaluate hydrodynamic changes after heat-setting. All pectin so-
lutions were formulated at 50 mM NaCl due to the polyelectrolyte
behavior of pectin; hred increases at dilute concentrations in the
absence of added salt due to long-range electrostatic repulsion
(Pals & Hermans, 1952). Ionic strength of the remaining samples
(6) was not adjusted. The WPI was adjusted to pH 6 due to precipita-
tion at pH 5.
The [h] of HMP (461 mL/g) falls in the established range for
citrus-derived HMP (Table 1) (Fishman, Gillespie, Sondney, & El-
 T.B. Wagoner, E.A. Foegeding / Food Hydrocolloids 63 (2017) 130e138
Table 1
Intrinsic viscosity of high-methoxyl pectin (HMP), whey protein isolate (WPI), and
4% WPIeHMP soluble complexes before and after heat-setting.
Sample pH [h] (mL/g)a
HMP (50 mM NaCl) 5.0 461.0 ± 8.8 a
WPI 6.0b 5.1 ± 0.6 d
SCs 5.0 93.6 ± 1.6 b
Heat-set SCsc 5.0 79.5 ± 4.2 c
Letters indicate significant differences among means within a column as determined
by Tukey's HSD at a ¼ 0.05.
a
Reported [h] means are averages based on the extrapolation of hrel/c and hsp/c to
zero concentration.
b
WPI was evaluated at pH 6.0 due to flocculation at pH 5.0.
c
Solutions were heat-set at 85
C for 25 min.
Atawy, 1991). An [h] of 5.1 mL/g for WPI is similar to other reported
values for WPI (Vardhanabhuti & Foegeding, 1999). This is greater
than reported values for belactoglobulin (3.7 mL/g) and the Ein-
stein hard sphere model for globular proteins (2.5 mL/g) (Ba ez,
Moro, Ballerini, Busti, & Delorenzi, 2011; Ross-Murphy, 1994). The
slight deviation from belg values can be attributed to the combi-
nation of proteins in WPI, notably the large percentage of GMP in
this sweet whey-derived ingredient. The [h] of unheated
WPIeHMP SCs was 93.6 mL/g. After heat-setting, SCs had a
significantly lower [h] of 79.5 mL/g, suggesting a reduced hydro-
dynamic volume after heat-setting. One explanation for the
reduced intrinsic viscosity is the formation of more tightly packed
(i.e., dense) complexes, which would be accompanied by a reduc-
tion in particle size and hydrodynamic volume. Similar behavior
has been reported for heated whey protein and pectin complexes,
where under certain conditions the mean particle size was lower
after heating above protein denaturation temperatures
(Kazmierski, Wicker, & Corredig, 2003; Krzeminski et al., 2014).
3.2. Particle size distribution
Intrinsic viscosity provided an estimate of hydrodynamic vol-
ume relative to mass but did not directly provide information about
particle size. In beverage applications, colloidal stability is most
important and is governed by the sedimentation rate of dispersed
particles. Although Stokes’ law is an ideal model representing an
isolated sphere at infinite dilution, it can be used as a simple sta-
bility model, where colloidal stability is predicted by small particle
size, minimal density difference between the particle and solvent,
or high continuous phase viscosity (Dickinson, 1992).
To evaluate the size distribution of SCs, a combination of tech-
niques e laser diffraction and dynamic light scattering (DLS) e
were used. These complementary techniques indirectly provide
size information based on scattering angle (laser diffraction) or
diffusion of particles due to Brownian motion (DLS). Additionally,
each technique favors a particular size class and implies certain
assumptions; thus, a combination of the two techniques is an
appropriate way to characterize the samples (Shekunov,
Chattopadhyay, Tong, & Chow, 2007). It is worth noting that un-
der our experimental conditions, both techniques assume a
spherical shape, thus giving a size approximation of an equivalent
hard sphere. Particle size distribution was described using number-
based D50 and D90 values, and D[4,3] volume mean diameter. D[4,3] is
a volume-weighted average and can be skewed by a small number
of large particles, as they occupy a greater volume than small par-
ticles; conversely, D50 and D90 values are not skewed by the pres-
ence of a few very large particles to the same extent (Xu, 2000).
Per laser diffraction, unheated WPIeHMP SCs had a monomodal
distribution with peaks ranging from 200 to 400 nm and a small,
trailing “shoulder” of particles in the 5e10 mm range (Fig. 1). An
133
increase in protein concentration from 1 to 6% did not affect the
shape of the size distribution, although the D50 and D[4,3] means
trended towards a larger diameter (Table 2). After heat-setting, the
SCs were generally smaller in D50, D90, and D[4,3] means at all
protein concentrations. As with the unheated SCs, a concentration
effect on particle diameter was observed.
The z-average diameter of unheated SCs ranged from 222 nm at
1% protein to 353 nm at 6% WPI (Table 3). Unheated SCs also tended
to be more variable in particle size (i.e., larger standard deviation),
which was similarly observed as greater polydispersity (i.e., wider
peak width) via laser diffraction. As the size population was poly-
disperse, particle size distributions were separated into three size
classes: 1e10 nm, 10e100 nm, and >100 nm. Of the unheated SCs
larger than 100 nm, mean diameter increased with protein con-
centration from 311 nm at 1% protein to 648 nm at 6% protein.
The z-average diameter of heat-set SCs increased from 257 nm
at 1% protein to 1046 nm at 6% protein (Table 3). Moreover, as
protein concentration increased the mass percentage decreased in
the smaller size classes and increased in the >100 nm size class.
This suggests an increased extent of aggregation at higher protein
concentration. This trend was previously reported for belg and
HMP complexes formed and heat-set at pH 4.75, where z-average
diameter increased from 350 nm (1.0% belg, 0.5% HMP) to 550 nm
(2.0% belg, 1.0% HMP) (Jones & McClements, 2010). Slight differ-
ences can be attributed to differences in polymer type and forma-
tion conditions, although these results generally agree with our DLS
results. Compared to heat-set SCs, unheated SCs had more mass in
the 1e10 nm size class (43e56%). After heat-setting, only 1e10% of
total mass fell in the 1e10 nm size class, with over 88% of mass in
the >100 nm size class.
A reduction in mean diameter after heat-setting would support
the decreased hydrodynamic volume observed from intrinsic vis-
cosity, whereas the increase in mean diameter observed using DLS
would support a greater degree of protein aggregation. In the case
of unheated SCs, D[4,3] means were approximately twice as large as
z-average diameter at all protein concentrations. This could be due
to assumptions on real and imaginary refractive indices of Mie
scattering theory. Conversely, the z-average diameter of heat-set
SCs was always larger than D[4,3] and the disparity increases with
protein concentration. The formation of more linear aggregates
during heat-setting would favor a larger calculated spherical
diameter.
One explanation for a transition towards a smaller diameter
after heat-setting is the core shell theory of structural rearrange-
ment (Jones & McClements, 2011; Peinado, Lesmes, Andre s, &
McClements, 2010; Santipanichwong, Suphantharika, Weiss, &
McClements, 2008). Protein denaturation at elevated tempera-
tures leads to weakened protein-polysaccharide electrostatic in-
teractions, disruption of hydrogen bonding, and complex
dissociation. The dissociated denatured proteins aggregate via
nucleation and growth to form microgels in the absence of poly-
saccharides (Bromley, Krebs, & Donald, 2006; Jones & McClements,
2010). As cationic patches reform on the aggregate exterior, elec-
trostatic interactions cause the anionic polysaccharide to attach on
the surface, limiting the extent of protein aggregation (Jones &
McClements, 2010). Thus, this reformed particle has a proposed
protein aggregate “core” and polysaccharide “shell.” The anionic
surface charge provides electrostatic stabilization. Additionally,
extension of the polysaccharide into the bulk solvent may provide
steric stabilization in the same way pectin stabilizes micellar casein
in acidic milk beverages (Tromp et al., 2004).
3.3. zePotential
The core shell theory would suggest greater surface charge (i.e.,
134 T.B. Wagoner, E.A. Foegeding / Food Hydrocolloids 63 (2017) 130e138

Fig. 1. Size distribution of unheated and heat-set WPIeHMP soluble complexes formed at pH 5. Percentage indicates protein concentration on a mass basis, at an 8:1 mass ratio of
protein to polysaccharide. Heat-set complexes were heated 25 min at 85
C.

Table 2 to 42.9 ± 0.4 at 6% WPI. These values are slightly more negative
Size characteristics of WPIeHMP soluble complexes (SCs) based on protein con- than the 32 mV reported for SCs (0.1% belg, 0.05% HMP) formed
centration as determined by laser diffraction.
and heat-set at pH 4.75 (Jones, Lesmes, Dubin, & McClements,
Protein (%) D50 (nm) D90 (nm) D[4,3] (nm) 2010). Unheated and heat-set SCs both exceeded the j30j mV
Unheated SCs 1.0 210 ± 54 bcd 1150 ± 432 ab 557 ± 241 ab threshold commonly used as a predictor of colloidal stability. The
4.0 271 ± 33 ab 1350 ± 234 a 664 ± 166 a greater magnitude of surface charge after heating agrees with the
5.0 243 ± 18 abc 1183 ± 140 ab 653 ± 248 a aforementioned core shell theory of structural rearrangement
6.0 300 ± 27 a 1436 ± 155 a 715 ± 100 a
during heating and would suggest that e at least in terms of surface
Heat-set SCs 1.0 172 ± 8 cd 493 ± 20 d 230 ± 4 c charge e SCs would exhibit greater colloidal stability after heat-
± ± ±
4.0 164 2 d 666 9 cd 273 2 c
setting. Furthermore, a greater zepotential magnitude after heat-
5.0 190 ± 3 cd 813 ± 15 bc 329 ± 7 bc
6.0 170 ± 4 cd 895 ± 14 cd 367 ± 6 bc
setting supports the core shell theory of stabilization during heat-
setting.
All complexes were formed at pH 5. Heat-set SCs were heated for 25 min at 85
C.
In regards to both size and zepotential, it is important to note
D50 and D90 represent the diameter that 50% or 90% of particles are smaller than.
D[4,3] values represent the volume mean average. Values are given ± standard de- that much of the research on complexation and heat-setting of
viation. Letters indicate significant differences among means within a column as whey protein/pectin complexes involves belg because it is a well-
determined by Tukey's HSD at a ¼ 0.05.

Table 3
Average particle size and mass percent of unheated and heat-set soluble complexes in various size classes.

Protein (%) z-Average diameter (nm) 1e10 nm 10e100 nm >100 nm

Mean diameter (nm) Mass (%) Mean diameter (nm) Mass (%) Mean diameter (nm) Mass (%)

1%, unheated 222 f 8.5 42.6 b 38.5 ab 0.4 311 e 57 c

4%, unheated 286 def 7.5 52.2 a 67.7 a 0.7 524 d 47 d
5%, unheated 316 de 8.2 51.3 a 61.2 ab 0.3 671 c 48 d
6%, unheated 353 d 6.1 56.3 a 58.5 ab 0.5 648 c 43 d

1%, heat-set 257 ef 11.3 9.9 c 23.3 b 1.4 303 e 89 b

4%, heat-set 585 c 6.2 6.3 cd 32.3 ab 0.5 692 c 93 ab
5%, heat-set 876 b 8.1 4.7 cd 900 b 95 ab
6%, heat-set 1046 a 3.6 1.1 d 1053 a 99 a

All complexes were formed at pH 5. Heat-set SCs were heated for 25 min at 85
C. Z-average diameter represents the intensity-weighted mean diameter. Mean diameter and
mass percentage are reported for three size classes: 1e10 nm, 10e100 nm, and >100 nm. Letters indicate significant differences among means within a column as determined
by Tukey's HSD at a ¼ 0.05.

electrostatic stabilization), which in addition to particle size is an
indicator of colloidal stability. Surface charge is commonly esti-
mated via zepotential, where a high magnitude of charge e either
positive or negative e is desired to attenuate flocculation. A j30j mV
threshold is often used as a guideline for predicting colloidal sta-
bility (Jacobs, Kayser, & Müller, 2000).
The zepotential of unheated SCs ranged from 33.6 ± 3.8 at 1%
protein to 39.4 ± 0.5 at 6% protein, suggesting a concentration
effect (Fig. 2). Heat-set SCs had a greater magnitude (i.e., more
negative) zepotential ranging from 38.9 ± 1.1 at 1% WPI
characterized model protein for mechanistic studies (Verheul,
Roefs, & de Kruif, 1998). However, from an application perspec-
tive, WPI is a more appropriate ingredient for beverages. In this
study, the WPI was derived from membrane processing and con-
tained 24.3% GMP. The SCs were formed at pH 5, which falls nears
the pI of belg (pH 5.3) and aelactalbumin (pH 4.8) (Swaisgood,
1982). However, GMP has a pI < 3.8 and would thus possess a net
negative charge at pH 5 (Bernal & Jelen, 1985). With a negative
charge, GMP would either, theoretically, interact with cationic
protein patches during structural rearrangement or remain
 T.B. Wagoner, E.A. Foegeding / Food Hydrocolloids 63 (2017) 130e138 135

Fig. 2. zePotential of whey protein isolate e high methoxyl pectin soluble complexes
before and after heat-setting. Complexes were formed at pH 5, and protein concen-
tration was varied at a protein to pectin ratio of 8:1 w/w. Letters indicate significant
differences among means as determined by Tukey’s HSD at a ¼ 0.05.
Fig. 3. Rheological profiles of dispersions of whey protein isolateehigh methoxyl
pectin soluble complexes. Solutions were formed at pH 5 and are either unheated
(solid line) or heat-set at 85
C for 25 min (dotted line). Percentage indicates protein
concentration at a constant 8:1 ratio of protein to pectin. Lines are drawn according to
the power law parameters presented in Table 2.

dispersed in the bulk continuous phase.

3.4. Apparent Viscosity
Variations in the size and shape of SCs would result in different
flow patterns of SC dispersions. Flow behavior is particularly
important due to the impact of viscosity on processing consider-
ations and consumer preferences (Thompson, Drake, Lopetcharat,
& Yates, 2004). Rheological behavior of protein-polysaccharide
coacervates and emulsions stabilized by SCs has been explored,
but fluid dispersions of protein-polysaccharide complexes have not
been well-characterized (Benichou, Aserin, Lutz, & Garti, 2007;
Neirynck et al., 2007; Stone & Nickerson, 2012; Wang, Lee, Wang,
& Huang, 2007; Weinbreck et al., 2004).
All samples showed at least some degree of shear-thinning
behavior and were thus modeled with Power Law parameters
(Table 4). Shear-thinning behavior is likely due to disruption of
interparticle linkages or to orientation of particles in the direction
of the shear field (Chen, Wen, Janmey, Crocker, & Yodh, 2010). At 1%
protein, both unheated and heat-set SCs were almost Newtonian in
nature (n > 0.98) with nearly identical flow profiles (Fig. 3). At 4e6%
protein, the heat-setting treatment resulted in a reduction in vis-
cosity over the measured shear rate range (0.1e200 s1). The drop
Table 4
Consistency and flow behavior coefficients of heat-set WPIeHMP SC dispersions.
Protein (%) K (mPa sn) n R2
in viscosity is in congruence with a smaller [h] and D[4,3] of heat-set
SCs, indicating that the viscosity change could simply be due to the
smaller hydrodynamic size/volume of the particles. An alternative
hypothesis is that unbound pectin dispersed in the continuous
phase becomes bound in particles (Jones & McClements, 2010).
Heat-set SCs exhibited more negative zepotential, suggesting that a
greater amount of pectin adsorbed to the surface of the particles.
Thus, less free pectin would decrease continuous phase viscosity.
This would also explain the lower [h] of heat-set SCs as the po-
tential contribution to bulk viscosity is lower when pectin is
adsorbed to the surface. Future studies comparing the amounts of
free/bound protein and pectin would be needed to test the veracity
of this hypothesis.
Solutions of both unheated and heat-set SCs trended towards
higher K and an increased degree of pseudoplasticity with
increasing protein concentration (Table 4, Fig. 3), which is expected
based on increased dispersed phase volume. Solutions containing
heat-set SCs at 5 and 6% protein exhibited lower K values than
unheated SCs. These values differ from other reported n (0.56) and
K (110 mPa sn) values of unheated SCs of WPI and enzymatically-
modified HMP formed at pH 6 (Lutz, Aserin, Portnoy, Gottlieb, &
Garti, 2009). The higher viscosity and greater degree of pseudo-
plasticity found by Lutz et al. (2009) could be attributed to varia-
tions in protein composition, pectin modification, pH, or to
differences in complex formation technique.
3.5. Application for soluble complexes in beverages

unheated SCs 1 1.90 f 0.98 a 0.98 As mentioned previously, one application of interest for heat-set
4 20.3 e 0.91 bc 0.99 SCs is in beverages with the benefit of increased thermal stability
5 39.5 c 0.89 cd 0.99 near the pI of whey proteins, where thermal processing produces
6 79.0 a 0.85 e 0.99
precipitates or gels (Wagoner et al., 2015). If thermal processing
heat-set SCs 1 1.92 f 0.99 a 0.95 produces sols, the primary concern for this application would be
4 16.8 e 0.92 b 0.99
colloidal stability, which would be predicted by a small particle size
5 31.9 d 0.87 de 0.99
6 68.0 b 0.86 e 0.98 per Stokes’ Law. This could be accomplished by combining heat-
setting and commercial sterility in one heating process, or using
Power law parameters K (consistency coefficient) and n (flow behavior coefficient)
of solutions containing unheated and heat-set WPIeHMP SCs as affected by protein
heat-set SCs as an ingredient prior to final formulation and thermal
concentration and heat-setting. Letters indicate significant differences among heating (Wagoner, Vardhanabhuti, & Foegeding, 2016). For the
means within a column as determined by Tukey's HSD at a ¼ 0.05. former option, small particles are only formed at a narrow pH range
136 T.B. Wagoner, E.A. Foegeding / Food Hydrocolloids 63 (2017) 130e138
close to the protein pI (Turgeon et al., 2007). With whey proteins
and pectin, this corresponds to pH 4.5e5.25 due to strong elec-
trostatic attraction and the tendency for polymers to remain
interacting during heating (Jones & McClements, 2011). For the
latter option, heat-setting may be a method of expanding the op-
timum range of pH and ionic strength (Jones & McClements, 2008;
Krzeminski et al., 2014). Additionally, the ratio of protein to poly-
saccharide could be altered, which has been shown to alter func-
tionality of SCs in emulsions (Li, Fang, Al-Assaf, Phillips, & Jiang,
2012). To evaluate the effects of pH on thermal stability of particles,
dispersions of heat-set SCs (4% protein) formed at pH 5 were
treated as model beverages and adjusted to pH 3, 4, 6, or 7 prior to a
UHT thermal process similar to commercial sterilization of near-
neutral pH protein beverages.
The particle size distribution of the model beverages after UHT
thermal processing is shown in Fig. 4. The control represents the
heat-set SCs at pH 5 that were not UHT processed. The size distri-
bution of the pH 4 model beverage was similar to that of the un-
heat processed control, with a D[4,3] of 303 nm compared to
273 nm for the control, suggesting a limited loss of stability
(Table 5). Stability at pH 4 is significant to beverage processing, as
beverages at pH values of 4.6 require a less severe heat process to
produce commercial sterility. At pH 6 and 7, the size distribution
shifted towards larger sizes, with D[4,3] values 2e3 times greater
than the control. Moreover, the pH 7 model beverage trended to-
wards slightly higher particle sizes than the beverage at pH 6,
although the means were not statistically distinct. Sedimentation
was not observed in these two treatments over the course of several
weeks, suggesting at least moderate stability; however, this would
need to be more rigorously evaluated. Previous reports indicate
that heat-set SCs (belg and HMP) are stable to pH adjustments
from pH 3e7 (Jones, Decker, & McClements, 2010). This would
indicate that the change in size distribution observed in this study
is due to the UHT process, not the pH adjustment step.
The beverage adjusted to pH 3, conversely, shifted towards a
bimodal distribution with peaks near 400 nm and 9 mm
(D[4,3] ¼ 2.86 mm) and sedimentation occurred rapidly. This
Fig. 4. Size distribution of heat-set whey protein isolate-high methoxyl pectin soluble
complexes (formed at pH 5 with 4% WPI and 8:1 ratio of protein to pectin). Solutions
were adjusted to pH 3, 4, 6 or 7 and thermally processed via UHT conditions (6 s at
141
C). The control represents SCs that were heat-set at pH 5 but not UHT processed.
Table 5
Particle size characteristics of heat-set WPI-HMP soluble complexes after pH
adjustment and UHT thermal processing.
pH D50 (nm) D90 (nm) D[4,3] (nm)
Control 164 ± 2c 666 ± 9 c 273 ± 2 b
3 423 ± 9a 8520 ± 1140 a 2860 ± 627 a
4 173 ± 7c 746 ± 51 c 303 ± 18 b
6 293 ± 61 b 1940 ± 560 b 712 ± 147 b
7 359 ± 74 ab 2310 ± 510 b 829 ± 158 b
Complexes were formed at pH 5 with 4% WPI and 8:1 ratio of protein to pectin,
adjusted to pH 3, 4, 6 or 7, and thermally processed via UHT conditions (6 s at
141
C). The control represents SCs that were heat-set at pH 5 but not UHT pro-
cessed. D50 and D90 represent the diameter that 50% or 90% of particles are smaller
than. D[4,3] values represent the volume mean average. Values are given ± standard
deviation. Letters indicate significant differences among means within a column as
determined by Tukey's HSD at a ¼ 0.05.
suggests aggregation of primary particles concomitant with the
lower magnitude of surface charge at pH 3 (Jones et al., 2009, 2010).
Galacturonic acid has a pKa near pH 3, so the surface of the SCs
would be less negatively charged at pH z pKa per the core shell
model (Whistler & BeMiller, 1993). This loss of electrostatic repul-
sion would induce flocculation and/or secondary aggregation of SCs
during UHT processing. Additionally, GMP has a pI < 3.8 (Bernal &
Jelen, 1985), and protonation at acidic pH could facilitate aggrega-
tion among SCs. The formation of particles >1 mm indicates that SCs
would not provide any benefit at pH 3, especially when considering
whey proteins alone and in beverages typically exhibit high ther-
mal stability in this pH range (Wagoner et al., 2015).
4. Conclusions
This study has shown that whey protein isolate and high-
methoxyl pectin can be used to generate soluble complexes at
concentrations up to 6% protein, which exceed FDA requirements
for a “good source” label claim. The average size of soluble com-
plexes increased as protein concentration was increased. Soluble
complexes can be heat-set at temperatures above the protein
denaturation temperature. Heat-setting was not detrimental to
stability of the soluble complexes, and moreover, was associated
with a transition to a narrower size distribution, reduced hydro-
dynamic volume, greater magnitude of surface charge, and a
reduction in apparent viscosity of the dispersion. These trends
would favor colloidal stability via a slower sedimentation rate per
Stokes’ Law and strong electrostatic repulsion. A model beverage at
pH 4 containing heat-set SCs at 4% w/v protein can be UHT pro-
cessed without a significant impact on particle size.
Acknowledgements
Support from the North Carolina Agricultural Research Service
and Glanbia Nutritionals is gratefully acknowledged. The use of
trade names in this publication does not imply endorsement by the
North Carolina Agricultural Search Service of the products named
nor criticism of similar ones not mentioned. The authors are
grateful for whey protein isolate and high-methoxyl pectin pro-
vided by Glanbia Nutritionals and CP Kelco, respectively. Assistance
and comments from Chris Daubert are gratefully appreciated.
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