Ultrasonics - Sonochemistry 70 (2021) 105340

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Ultrasonics - Sonochemistry

journal homepage: www.elsevier.com/locate/ultson

High-intensity ultrasound pretreatment influence on whey protein isolate and its T

use on complex coacervation with kappa carrageenan: Evaluation of selected
functional properties
Sara A. Vargasa, R.J. Delgado-Macuila, H. Ruiz-Espinosac, M. Rojas-Lópeza, G.G. Amador-
Espejob,1
a
Instituto Politécnico Nacional, Centro de Investigación en Biotecnología Aplicada, México, Ex-Hacienda San Juan Molino Carretera Estatal Tecuexcomac-Tepetitla Km 1.
5, 90700 Tlaxcala Mexico
b
CONACYT-Centro de Investigación en Biotecnología Aplicada IPN, México, Ex-Hacienda San Juan Molino Carretera Estatal Tecuexcomac-Tepetitla Km 1.5, 90700 Tlaxcala,
Mexico
c
Benemérita Universidad Autónoma de Puebla, México, Facultad de Ingeniería Química, Colegio de Ingeniería en Alimentos, 18 Sur y Avenida San Claudio, 72570 Puebla,
Mexico

ARTICLEINFO ABSTRACT
Keywords: The aim of this work was to evaluate the influence of high-intensity ultrasound (HIUS) treatment on whey protein isolate
Complex coacervation (WPI) molecular structure as a previous step for complex coacervation (CC) with kappa-carrageenan (KC) and its influence on
High-intensity ultrasound CC functional properties. Protein suspension of WPI (1% w/w) was treated with an ultrasound probe (24 kHz, 2 and 4 min, at
Whey protein isolate Kappa 50 and 100% amplitude), non HIUS pretreated WPI was used as a control. Coacervation was achieved by mixing WPI and KC
carrageenan dispersions (10 min). Time and amplitude of the sonication treatment had a direct effect on the molecular structure of the
Functional properties protein, FTIR-ATR analysis detected changes on pretreated WPI secondary structure (1600–1700 cm −1) after sonication. CC
electrostatic interactions were detected between WPI positive regions, KC sulfate group (1200–1260 cm −1), and the
anhydrous oxygen of the 3,6 anhydro-D-galactose (940–1066 cm −1) with a partial negative charge. After ultrasound
treatment, a progressive decrease in WPI particle size (nm) was detected. Rheology results showed pseudoplastic behavior
for both, KC and CC, with a significant change on the viscosity level. Further, volume increment, stability, and expansion
percentages of CC foams were improved using WPI sonicated. Besides, HIUS treatment had a positive effect on the
emulsifying properties of the CC, increasing the time emulsion stability percentage. HIUS proved to be an efficient tool to
improve functional properties in WPI-KC CC.

1. Introduction Meanwhile, Whey protein isolate (WPI) presents important

Complex coacervation occurs when two compounds with
opposite net charges interact on a liquid media, producing a
spontaneous separation phase as a result of the complexation,
producing two phases, one rich in solvent and the other rich in
colloidal aggregates or coacervates [1,2]. This process depends on
many environmental and controllable factors like pH, ionic
strength, biopolymers nature, mixing weight ratio, conformational
molecular changes, molecular weight, density charge and
biopolymer flexibility [3]. Nowadays, complex coacervation
systems (CC) from proteins and polysaccharides, has been more
studied in order to obtain handmade complexes, through
controlling environmental factors. Recently, they have been used
in food processing because both biopolymers have specific
functional properties that may increase when they interact.
Received 25 June 2020; Received in revised form 16 August 2020; Accepted 3 September 2020
Available online 09 September 2020
1350-4177/ © 2020 Elsevier B.V. All rights reserved.
functional properties such as gelling, foaming, thickening, water
binding and emulsifying [4]. Meanwhile, kappa Carrageenan (KC)
could be used for increasing the viscosity in aqueous systems and
also as a gelling agent by its rheological properties. Several studies
have reported the increase in functional properties by CC use such
as emulsifying, foaming, water holding capacity, among others
[3,5–7].
Kappa Carrageenan (KC) is a sulfated polysaccharide extracted
from edible red algae, it is composed by D-galactose 4-sulfate and
3, 6-anhydro-galactopyranose units bounded by α-1,3 and β-1,4
glyosidic bond [8–10]. Since KC is a sulfated polysaccharide, it has
a negative net charge in a wide pH range, which allows it to
interact with positively charged compounds like WPI under its
isoelectric point where the protein has a positive net charge [11–
13].

1 Corresponding author.
E-mail address: ggamadores@conacyt.mx (G.G. Amador-Espejo).

https://doi.org/10.1016/j.ultsonch.2020.105340
S.A. Vargas, et al.
Whey protein isolate (WPI) is obtained by ultrafiltration
process from milk whey, which is a byproduct in cheese making
process and has become popular as a food additive because of
their nutritional, functional and active properties [4,14,15]. It is
mainly composed by proteins such as β-lactoglobulin (β-LG) and α-
lactalbumin (α-LA) along with small amounts of
glycomacropeptide (GMP), immunoglobulins (Igs), bovine serum
albumin (BSA), lactoferrin (LF), lactoperoxidase (LP), proteose
peptone (PP), among others. β-LG, the main protein in whey
(~60%) conformed by a “calyx” or globulet-like structure, with the
capacity to bind with numerous hydrophobic and amphiphilic
ligands [16]. To improve the accessibility to reactive groups, it is
necessary to previously denature and/or unfold globular compact
structures formed mainly by albumins and globulins fractions
(ionic, hydrophobic, -SH groups) and to promote the interaction
with other polymers, such as complex coacervation [17].
High-Intensity Ultrasound (HIUS) is a non-thermal, green
technology that has been used to induce conformational changes
in proteins affecting physicochemical properties of a number of
protein sources including whey protein isolate/concentrate, where
the main changes occur in surface hydrophobicity, viscosity,
particle size, and formation of protein–protein aggregates [18–22].
Ultrasound is an acoustic wave, imperceptible for the human
audition with a frequency higher than 20 kHz. This technology
particularly employs frequencies ranging from 100 kHz to 1 MHz,
its fundamental work process is attributed to the ultrasonic
cavitation, which refers to the rapid formation and collapse of gas
bubbles in a liquid media, generating pressure and temperature
increases at specific points by very short periods of time
(milliseconds), producing changes on the surrounding molecules in
the specific places where the bubbles collapse due to pressure
differentials [23]. Due to cavitation effects, ultrasonic treatment
can increase the probability of contact between the
proteinpolysaccharide complex and bioactive compounds, and
increase the stability of complex coacervation [24].
Many studies have reported the study of CC from proteins and
polysaccharides but only a few studies have investigated the
influence of ultrasonication as a previous step for induced protein
conformational changes. HIUS has proven useful on the
improvement of CC functional properties like emulsion
stabilization on whey protein concentratepectin complexes [25],
and also to encapsulate polyphenolic compounds such as
resveratrol [24]. One of the main limitations between
protein/polysaccharide interactions is their molecular structure
and its ability to interact. In this sense, the use of HIUS may modify
the structure of biopolymers and consequently, their technological
properties [17]. Based on the many studies, it is hypothesized that
ultrasound treatment could induce modifications of the physical
and functional properties of WPI-KC complex coacervation, which
could be useful in the development of CC systems with improved
functional properties. For this, the aim of this research was to
evaluate the effect of HIUS on the structural, physicochemical and
functional properties of WPI-KC CC, using the ultrasound
treatment as a previous step on the WPI conformational
modification.
2. Materials and methods
2.1. Materials
Refined KC (PEISA HX Gel RP 1000, México) and WPI (BiPro,
USA) were employed. The chemical composition of the WPI (w/w)
was protein: 91.0%, calcium: 0.05%, Sodium: 0.85%. Other
chemicals used in this study were analytical and reagent grade
purchased through Sigma- Aldrich (Sigma, St. Louis, MO, USA).
Ultrasonics - Sonochemistry 70 (2021) 105340
2.2. Preparation of stock dispersions
WPI (1% w/w) and KC (1% w/w) stock dispersions were prepared
by dispersing in deionized water with regular stirring for 24 h at 25
°C to guarantee complete hydration of biopolymers in order to use
on the following day. Sodium benzoate (0.1% w/w) was added to
prevent microbial growth.
2.3. Ultrasonic treatment of WPI dispersion
For ultrasound treatments, a HIUS probe equipment (Hielscher
UP400S, Germany, power 400 W, frequency 24 kHz) was used.
Aliquots of 100 mL of WPI stock dispersion were placed in a 150
mL flat-bottomed conical reactor, then a 22 mm diameter titanium
probe was submerged 3 cm in the liquid sample during
ultrasonication. The ultrasound irradiation was produced directly
from the tip under continuous mode. Each aliquot was treated
separately. The following HIUS treatments for WPI were applied:
Amplitude: 50 and 100% for 2 and 4 min. During ultrasound
treatments, the temperature was controlled by a recycling cool
bath at 25 ± 2 °C. The actual power delivered to the dispersed
sample was determined by the calorimetric method, based on the
equations presented by Kentish [26].
2.4. Protein surface hydrophobicity measurements
Surface hydrophobicity (S0) of the WPI was determined by
fluorometric test. As Chandrapala et al. [27] suggested, a stock
solution of 1anilinonaphthalene-8-sulphonate (ANS) 8 mM and
stock solutions of protein in phosphate buffer 0.1 M, pH 7 were
prepared using concentration ranges from 0.005 to 0.025% w/w.
20 µL of the ANS solution was added to 4 mL of each diluted
protein solution, vortexed and kept in the dark, then from lowest
to highest concentration of protein, the relative fluorescence
intensity (RFI) of each solution was measured using a fluorimeter
(Ocean Optics USB4000, USA) at an excitation and emission
wavelength of 390/470 nm, respectively. Net RFI was determined
by subtracting the intensity of the control (buffer pH 7.0), from the
intensity of the protein solution with ANS, at each protein
concentration. The degree of surface hydrophobicity (S 0) was
expressed as the initial slope: fluorescence intensity/ protein
concentration (%), and was calculated from the fluorescence
intensity vs. protein concentration plot, using the linear least
squares regression analysis through Microsoft Excel 2016
software.
2.5. Particle size (PS) distribution and zeta potential
determination
The PS distribution and Zeta potential (ζ, mV) of WPI samples
were determined after ultrasound treatments, through a
Microtrac DSL device (Nanotrac Wave II, USA), 1 mL of the
dispersed sample was used. All measurements were carried out by
triplicate at 25 °C.
2.6. WPI-KC complex coacervation
2.6.1. Turbidimetric analysis at different pHs
Different mixtures of WPI and KC were prepared by mixing the
stock solutions at 1:1 (w/w) WPI: KC mixing ratio and a total
biopolymer concentration of 2% (w/w). The mixtures were
acidified gradually by the addition of 0.1 M HCl (pH range of 3–7).
The optical density (OD) of stock solutions of WPI, KC and WPI-KC
mixtures were assessed over time during the slow acidification
using a UV–Vis spectrophotometer (Thermo Sci Spectroscopy 14-
2
S.A. Vargas, et al.
386-441, USA) at 600 nm, using plastic cuvettes (1 cm path length).
Deionized water was used as a blank reference. The optimum pH
for coacervation corresponds to the pH value at which the highest
optical density was observed, this value as well as the critical pH
intervals in which there are coacervation stage transitions (pH opt:
maximum optical density, pHⱷ1: formation of insoluble complexes,
and pHⱷ2: dissolution of complexes) were determined graphically
from the curve [11,12]. All measurements were carried out by
triplicate.
2.6.2. Fourier transform infrared spectroscopy (FTIR-ATR)
WPI, KC and WPI-KC samples were analyzed by Fourier
transform infrared spectrometry (FTIR), 3 µL of each sample were
used. For this determination, FTIR spectrometer, (Bruker Vertex
70, USA) ATR mode (with a Platinum ATR accessory), in the
infrared region between 4000 and 400 cm −1 was used. All the data
were analyzed using Origin 8.0 (OriginLab, Northampton, MA,
USA). The modification on the secondary structure of WPI by HIUS
treatment was determined from amide I region (1600–1700 cm −1)
second derivative deconvolution and peak fitting (R 2 > 0.98),
employing the assignment of amide I band positions to secondary
structure [28].
2.6.3. Optical microscopy
To describe the WPI-KC complex coacervation and foams
structure an optical microscopy technique was employed at 40x
and 10x, respectively. Optical microscopy images were obtained
using an optical microscope (Carl Zeiss 467230, Germany).
Microscope analysis was carried out using the IMAGEJ (Java Image
Processing Program software, USA).
2.7. Functional properties of complex coacervates
2.7.1. Rheological behavior
KC, WPI, and CC rheological properties were determined using
a Brookfield rheometer (RST-CPS, USA) with temperature control,
coupled to the RCT-50 measuring steel cone at 25 °C, and rotation
speed: 2000 1/s. The data on the rheological behavior were
adjusted to the Power Law (Eq. 1) and Herschel–Bulkley (Eq. 2)
model using the following equations:
T = K * γn
T = T0 + (K * γn) (2) where γ is the shear speed, K and n are
constants, the viscous index and the consistency index
respectively, T represents the shear stress, and T 0 is the shear
stress at time 0.
2.7.2. Foaming capacity
Volume increase, foaming stability and expansion were
calculated according to the equations used by Silva Diniz et al.
[29]. To evaluate the foaming capacity, 70 mL of the WPI
dispersions and 70 mL of the complex coacervates dispersions
were used. Each sample was homogenized in a 150 mL beaker
with a homogenizer (Dremel 220, USA) for 60 s at 11,000 rpm. The
total volume and foam volume were measured immediately after
homogenization. The percentage of volume increase or overrun
was calculated employing Eq. 3:
VI (%) = [(PV2-PV1)/ PV1] *100 (3) where VI (%), represents the
volume increase, PV2 is the protein suspension volume after
agitation, PV1 protein suspension volume before agitation. The
variation in foam volume at 25 °C was measured immediately after
stirring, at intervals of 5 min for 1 h.
The percentage of foaming stability (FS%) and foam expansion
capacity (FE%), were evaluated using Eq. 4 and 5:
Ultrasonics - Sonochemistry 70 (2021) 105340
FS(%) = (Vff/Vf0)*100 (4) FE(%) = (Vf0/Vd)*100 (5) where
FS (%) is the foam stability percentage, V f0, is the foam volume at
time zero and Vff is the foam volume after time t, FE (%) is the
percentage of foam expansion and V d is the initial volume of the
dispersion.
2.7.3. Solubility determination
CC were lyophilized in freeze dryer (Labconco 7740021, USA).
Lyophilized CC powders were dispersed (1% w/v) in deionized
water at 25 °C and stirred at 110 rpm for 30 min. Then, the
samples were centrifuged at 4000 rpm for 5 min. Aliquots of each
supernatant were taken with volumetric pipettes and transferred
to previously weighed aluminum dishes, for dried to constant
weight in an incubator at 100 °C. The solubility was calculated
from the difference in weight [30].
2.7.4. Emulsifying properties
Oil-in-water emulsions were prepared at room temperature by
adding 12 mL sunflower oil into 48 mL of 3% CC dispersions (w/v).
The mixtures were homogenized in 150 mL beaker with a
homogenizer (Dremel 220, USA) for 90 s at 11,000 rpm to form
emulsions [31,32]. The emulsifying activity index (EAI) and
emulsion stability index (ESI) were determined by the turbid
metric technique as previously described [32,33]. Immediately
after emulsion formation, a 10 µL aliquot was transferred into 5.0
mL of pH-adjusted water containing 0.1% sodium dodecyl sulfate
(SDS), vortexed for 10 s. The absorbance was measured at 500 nm
using an UV– Vis spectrophotometer (Thermo Sci Spectroscopy 14-
386-441, USA). A second 10 µL aliquot of the emulsion was taken
10 min later, following the same procedure. EAI and ESI values
were determined by using the equations (7) and (8) [32,33].
EAI (m2/g) = (2*2.303*A0*N) / (c*ϕ*10000)
ESI (min) = (A0/ ΔA)*t (8) where A0 is the absorbance at 500 nm of
the diluted solution immediately after emulsion formation, N is
the dilution factor (500 x), c is the weight of protein per volume
before the emulsion was formed (g/ mL), ⱷ is the oil volume
fraction of the emulsion, ΔA is the change in absorbance between
0 and 10 min (A0-A10) and t is the time interval of 10 min.
2.7.5. Gelation properties
To induce thermal gelation, CC dispersions were thermally
treated in a water bath at 95 °C for 10 min, then gelation was
carried out at room temperature [34].
2.8. Statistical analysis
All experiments were carried out at least by duplicate. The
results were reported as averages with standard deviations and
were submitted to variance analysis (ANOVA) and Tukey test was
applied to evaluate significant differences among the mean values
(p < 0.05).
3. Results and discussion
3.1. Effect of sonication on the physicochemical properties of WPI
The different HIUS treatments on WPI and the actual power
delivered to the samples were: a) 50% amplitude, 2 min,
calorimetric power = 33.48 ± 2.0 W; b) 50% amplitude, 4 min,
calorimetric power = 38.12 ± 3.0 W; c) 100% amplitude, 2 min,
calorimetric power 83.18 ± 6.0 W and d) 100% amplitude, 4 min,
calorimetric power = 67.05 ± 3.62 W. The energy consumption for
the treatments 50% and 100% amplitude was 0.2 kWh/L and 0.4
kWh/L, respectively.
3
S.A. Vargas, et al.
The effectiveness of the sonication treatments, was evaluated
by the changes on WPI surface hydrophobicity, particle size, Zeta
potential and FTIR-ATR amide I (1600–1700 cm −1) second
derivative deconvolution.
3.1.1. Surface hydrophobicity measurements
It is well known the importance of protein structure and
hydrophobic interactions for the stability, conformation and
functional properties of proteins (emulsifying and foaming
capacity). Due to the macromolecular structure of proteins, the
surface hydrophobicity has more influence on functionality than
the total hydrophobicity [27]. In general terms, protein regions
associated with high hydrophobicity are
Table 1
WPI Surface hydrophobicity measurements.
HIUS treatment on WPI Surface Hydrophobicity Index (S Table 2
– 471.78 ± 1.97b
50%A, 2 min
437.5 ± 12.87b,c
50%A, 4 min 385.6 ± 28.0c 548.5 ±
100%A, 2 min 14.6a
100% A, 4 min 502.1 ± 14.9a,b
Different letters in the same column present significant difference (p < 0.05).
directed towards the interior of the protein and not exposed to
the surface, with the ultrasonic treatment, the hydrophobic
groups of the proteins initially localized in the interior of the
molecule are exposed to the more polar surrounding environment
[35,36]. The surface hydrophobicity of WPI dispersions as a
function of sonication treatment are present in Table 1. WPI
surface hydrophobicity decreased after 50% amplitude
treatments, which is a sing of protein aggregation, the partially
denaturized proteins might cause more extensive bonding, which
in turn protects the hydrophobic regions of the proteins [27], as
the amplitude of the sonication treatment increased (100%
amplitude), the surface hydrophobicity increases, high-intensity
ultrasound treatment (HIUS) induced structural changes in
proteins that were associated with partial scission of
intermolecular hydrophobic interactions due to the mechanical
effects of cavitation [22]. Ultrasound can modify the conformation
of the whey protein through affecting hydrogen bonds and
hydrophobic interactions, by acoustic and hydrodynamic
cavitation [37], increasing surface hydrophobicity, as had been
demonstrated in several studies [36,37]. These results also
support the trends observed in secondary structure data (Table 3),
where conformational changes in the protein structure induced by
ultrasound treatment were observed.
3.1.2. Particle size distribution
Particle size distribution (Table 2) showed that ultrasonic
treatments applied have a significant influence on particle size
reduction (p < 0.05) compared to the WPI untreated. In general, as
the treatment amplitude increases (from 50 to 100%) the particle
size decreases, which had been reported in other studies [22,35].
Ultrasound treatment has been reported to have significant
effects on the particle size reduction of various proteins
suspensions, including whey proteins; the main mechanisms
involved are ultrasonic cavitation, microstreaming and turbulent
forces that can reduce particle size of large macromolecules and
disrupt protein aggregates, increasing particle surface area and
Table 3
Secondary WPI structure estimated from deconvolution of amide I spectra, relative peak area (%).
Secondary structure relative peak area (%)
HIUS treatment on WPI R2 α- helix
a c
– 0.990 18.7 ± 0.31
50%A, 2 min 0.992 11.88 ± 0.09b
50%A, 4 min 0.986 5.43 ± 0.13d
100%A, 2 min 0.974 9.63 ± 0.12c
100% A, 4 min 0.971 8.83 ± 0.46c
For the same sample, groups with different capital letters have a significant statistical difference (p < 0.05).
Ultrasonics - Sonochemistry 70 (2021) 105340
improving particle interactions [36,38]. The particle size
decreasing after ultrasonic treatment also generate significant
structural changes disrupting electrostatic interactions such as,
hydrogen bonds, van der Waals forces, partial cleavage of
intermolecular hydrophobic interactions, rather than peptide or
disulphide bonds [22], leading improvements of emulsifying and
foaming properties without modifications in the primary structure
and the molecular weight, because the energy is insufficient to
hydrolyze the peptide bond [37].
3.1.3. Zeta potential
Through zeta-potential it is possible to known the stability of
colloidal systems by measuring the electrophoretic properties of
the
WPI particle size and zeta-potential after HIUS treatment.
HIUS treatment on WPI Particle size, Mz (nm) Zeta Potential
a
– 287.75 ± 3.75 14.90 ± 0.56a
50%A, 2 min 254.05 ± 5.30b 15.65 ± 0.64a
50%A, 4 min 223.95 ± 0.636c 15.70 ± 0.42a
100%A, 2 min 217.25 ± 2.47c 15.30 ± 0.00a
100% A, 4 min 190.60 ± 10.75d 15.60 ± 0.28a
Different letters in the same column present significant difference (p < 0.05).
colloidal particles. As an important parameter, surface charge of
particles can affect their solubility and interactions with other
particles [32,39]. The effects of ultrasound treatment on zeta-
potential are shown in Table 2. As expected [11], the untreated
whey protein particles presented positive zeta-potential value
(cationic character) 14.9 ± 0.56 mV at pH lower (pH = 3) than its
isoelectric point (IEP: 5–5.2) [13,40,41]. At this pH, WPI dispersion
exhibited the maximum cationic character in a zeta-potential value
[11]. After sonication treatments, although there was an increase
in zeta potential, this increase was not statistically modified (p <
0.05), maintaining the cationic character of WPI protein, which is
important characteristic for complex coacervation.
3.1.4. Fourier transform infrared spectroscopy (FTIR-ATR),
deconvolutionof WPI amide I second derivative
Several studies have analyzed the protein structural changes
after ultrasound treatment [23,32,37]. Conformational changes on
WPI are the result of partial splitting, inhibition of aggregate
formation or protein–protein interactions [42]. Proteins stability
depends on their ability to form the maximum number of
hydrogen bonds, complemented by Van der Waals interactions,
derived from the hydrogen bonds between the carbonyl oxygen
and the amine hydrogen of the peptide bonds, however, changes
in the availability of –OH groups due to HIUS treatment can
disrupt the balance causing structural changes [14]. For the
formation of secondary structure elements, a specific type of
electrostatic interactions, the hydrogen bonds, are often claimed
as the main stabilizing force; an hydrogen atom that is covalently
bound to a nitrogen, gets in proximity of, and is therefore
attracted by, an electronegative oxygen [43]. The stable secondary
structure elements then further develop interactions between
themselves and form a tertiary structure which is the folding of
secondary structure into distant arrangements know as domains
[44]. In this sense, secondary structure of the proteins can be used
to predict the tertiary structure [45]. In this work, FTIR-ATR was
β-sheets β- turns Disordered
c
33.55 ± 0.07 31.47 ± 0.63 16.28 ± 0.26b
36.35 ± 0.29b,c 34.88 ± 0.14b 16.89 ± 0.34b
39.92 ± 2.72b 42.85 ± 2.56a 11.8 ± 0.29c
4
46.29 ± 0.23a 24.4 ± 0.06c 19.68 ± 0.41a
48.78 ± 0.19a 23.28 ± 0.4c 19.1 ± 0.67a
S.A. Vargas, et al.
employed to characterize molecular protein secondary structure
as a function of HIUS treatment conditions, which is commonly
based on the amide I band (C = O, N = O vibrations) analysis
(1700–1600 cm−1). For this purpose, second derivative of this
region was obtained and then de-convoluted and peak fitted (R 2 >
0.97). The bands analyzed were those that correspond to the
contributions of β-sheets (1617–1642 cm −1), β-turns (1672–1691
cm−1), α-helix (1655–1662 cm−1) and random structures (1646–
1652 cm−1) [46]. The configuration of the secondary structures has
a great influence on the stability of the protein structure [43], in
order to quantify the variation of the corresponding contributions,
areas under the curves were calculated and presented in Table 3.
Infrared studies have suggested that the β-Lg (~60% of WPI)
secondary structure consists of 10 to 15% of α-helices, ~50% of β-
sheets and ~20% of turns, the remainder ~15% represents amino
acid residues in a disorganized non-repetitive arrangement
without a well-defined structure [47]. The modification on
secondary structure from HIUS treatments (Table 3), include
significant (p < 0.05) conformational changes in all the treatments;
however, treatments applied at 100% amplitude are those that in
general terms included a decrease on αhelix (1655–1662 cm −1), an
increase on β sheets (1617–1642 cm −1), turns (1672–1691 cm−1)
and disordered structures (1646–1662 cm −1). The reduction in α-
helix content might correspond to the partial unfolding of the α-
helical region that is caused by ultrasonic cavitation, because
ultrasound treatment can reduce the number of intramolecular
bonds, by the other hand, the increase in random coils can be
attributed to the transformation of β-turns into random coils [38].
Additionally, when a protein presented an increase in disordered
structures, it is believed that it has undergone a greater
conformational changes [46]. The increase in β-sheet structure
contributed to the exposure of the hydrophobic regions of the
protein [38]. Wang et al. [42] suggest that there is a positive
correlation between β-sheet-random coil content and surface
hydrophobicity, the higher β-sheet and random coil content, the
bigger value of surface hydrophobicity, which is in concordance
with the results obtained in the present work (Tables 1 and 3),
being the most significant results those corresponding to 100%
amplitude, 2 min treatment. The most probable reason for this
behavior is that the content of α-helix in protein is low and the
protein molecular structure is relatively lost when the β-sheet and
random coil content is high. Besides, the hydrophobic sites in the
protein are more largely exposed, increasing the surface
hydrophobicity by unmasking internal hydrophobic regions as a
consequence of the unfolding of the proteins, this influence the
protein functional properties [36,42].
3.2. Complex coacervation
3.2.1. Turbidimetric analysis at different pHs
Protein ionization due to amine and carboxyl groups is
determined by pH, its charge largely determines electrostatic
interactions with other biopolymers, including polysaccharides. It
is recognize that, the major driving force involved in complex
coacervation between protein and anionic polysaccharides, such
as KC, in solution is the electrostatic interaction [2,48], thus pH is a
key factor for complex coacervation. To identify the optimal pH,
the optical density (OD) curves were obtained as a function of
changes in pH maintaining the biopolymers ratio (Fig. 1), KC and
WPI were used as a control.
KC is a sulfated polygalactan negatively charged in a wide pH
range due to the presence of sulfate groups in its molecular
structure [8,9,10], regardless of the pH level, no significant
changes in the OD of the KC titration curve were observed.
However, in the case of WPI, a significant increase in the turbidity
level (pH ~ 5) was detected, this increase corresponds to the
Ultrasonics - Sonochemistry 70 (2021) 105340
average isoelectric point (IEP) of the WPI, which is in agreement
with previous reports (WPI, IEP = 5.2) [41,49]. WPI solutions tend
to be stable over a wide pH range except close to its IEP, where
the dimer- monomer equilibrium of β-Lg is shifted toward the
monomer, changing the structure of the aggregates considerably
[50], as a consequence, maximum turbidity level is achieved. The
pH
lower or higher that the IEP resulted in a steady decrease in the
turbidity [41], pH levels below the IEP confer a protein positive net
charge, while pH levels above the isoelectric point confer a
negative net charge [11,40].
By the other hand, as complex coacervation depends on
electrostatic interactions and therefore on pH, there were
different stages when the process was carried out. Fig. 1 shows
the characteristic transition stages observed by changes in
turbidity of the WPI-KC complex. In the first stage, called pH 0, the
presence of co-soluble biopolymers was exhibited, where non-
electrostatic interactions occurred between them (pH 0 ~ 7). At this
stage, the optical density level was the minimum, the interaction
between the biopolymers does not occur because both materials
presented a negative charge and repelled each other
electrostatically. The second stage, pHφ1, occurred when the pH
was reduced, at this point (pHφ1 ~ 6) the interaction between both
biopolymers begins to increase, as the OD increase, and the
insoluble complexes are formed. Subsequently, as the pH
decreases, the complexes continued to grow in size until the
system reached the third stage, the optimum formation point,
called pHopt, which was evidenced by a maximum in OD (pH opt ~
4.5), where maximum complex formation was achieved.
Subsequently, a decrease in OD was observed (pHϕ2 ~ 4) indicating
the dissociation caused by protonation of reactive groups,
resulting in less interactions and the dissociation of the complex
structure [11,51]. Complex coacervation, protein–polysaccharide,
could occur when the pH of the mixture was lower than the IEP of
the protein. At this pH, the protein possessed a net positive charge
whereas the polysaccharide still possessed a negative charge [2].
From this experiment, it was possible to determine that the
optimum pH for coacervation was reached when CC system pH ~
4.5. This pH media level was achieved by mixing WPIKC, when WPI
(pH 3) had a positive net charge and KC (pH 7) presented a
negative net charge, allowing that both biopolymers interacted
electrostatically.
On the other hand, OD of sonicated WPI and HIUS WPI- KC
complex coacervates was determined in order to know the
influence of the ultrasonic treatment. KC and WPI, CC without
treatment were used as a control, these results are shown in Table
4.
It is well known that ultrasonic treatment could modify the
protein molecular structure, this change can be evaluated by
5
S.A. Vargas, et al. Ultrasonics - Sonochemistry 70 (2021) 105340
changes in solution turbidity. In this sense, a significant increase in OD level (p < 0.05) was detected in 50% amplitude (33–38 W)
treatments.

6
S.A. Vargas, et al. Ultrasonics - Sonochemistry 70 (2021) 105340

(d) (e)

Fig. 1. Optical density as a function of pH for: WPI, KC, WPI-KC CC.

Table 4
(f)
WPI, KC and CC Optical density (600 nm). (g)
HIUS treatment OD (600 nm)

WPI CC KC

– 0.120 ± 0.013b 2.859 ± 0.027a

0.229 ± 0.116
50%A, 2 min 0.283 ± 0.015a 2.989 ± 0.147a 50%A, 4 min 0.262 ± 0.021a 2.838 ± 0.123a 100%A, 2 min
0.117 ± 0.007b 2.561 ± 0.325a
100% A, 4 min 0.051 ± 0.008c 2.542 ± 0.344a

Different letters in the same column present significant difference (p < 0.05).
Fig. 2. Optical microscopy (40x), morphology of: (a) WPI, (b) KC, (c) WPI-KC CC and WPI HIUS- KC CC: (d) 50% A, 2 min, (e) 50% A, 4 min, (f) 100% A, 2 min, (g)
100% A, 4 min.
Martini, Potter, and Walsh [52] suggest that ultrasonic low energy
treatments could increase turbidity level, although the mechanism
that leads an increase in the OD of protein samples is not entirely
clear. A possible mechanism may involve changes on the tertiary
structure of whey proteins and/or the minimization of protein–
protein interactions that leads to aggregation and as a
consequence an increase in turbidity. Contrarily, as the ultrasonic
treatment increases, 100% amplitude (67–83 W) there was a
significant decrease in the level of OD. This decrease could be
based on the scission of the main chain, due to ultrasonic
cavitation, also resulting in a decrease on the particle size of the
WPI [39], which is in concordance with the results of particle size
(Table 2).
CC samples did not present significant differences (p < 0.05)
with respect to the control, so it is inferred that protein ultrasonic
pretreatment does not have influence on the OD level of the
coacervates systems.
WPI, KC, and CC without HIUS treatment were used as a
control.
bigger size (11–16 μm) resulting from the union of several
individual complex coacervates (CC) [11].
The coacervated phase can be considered as a network of
interconnected intrapolymeric complexes with contacts that are
transiently stabilized by the association with protein molecules or
as a heterogeneous network containing entangled and cross-
linked protein and polysaccharide rich phases, both the protein
and the polysaccharide are found at the interface of the
coacervates, and its structural conformation influences
coacervation [1]. Further, WPI HIUS pre-treatment had an
influence in complex coacervation (Fig. 2d-2g). In this case, an
increase in the HIUS treatment amplitude presented
conformational changes in protein structure, which lead to
different complexation with a trending to disintegrate the overall
macro-complex (coacervatecoacervate) into individual coacervate
forms [11].

3.2.2. Optical microscopy

The morphology of individual biopolymers and complex
coacervates is illustrated in Fig. 2 (a-k). WPI (Fig. 2a) showed
spherical shape (0.2–1 μm), typical of globular proteins, where
nonpolar groups were oriented inside and polar groups to the
outside of the molecule [53]. The morphology of KC (Fig. 2b)
corresponds to their molecular structure, it has a double helix
conformation where the helical linear portions can be associated
to form a three-dimensional network [54].
On the other hand, complex coacervates control (Fig. 2c) had a
completely different morphology than that of individual
biopolymers, with spherical and defined shapes (2–3 μm), typical
of coacervates, resulted from the interaction between both
biopolymers. Also it is possible to observe macro complexes of Fig. 3. FTIR-ATR spectra (4000–400 cm−1) of individual biopolymers, WPI, KC
and WPI-KC CC.

7
S.A. Vargas, et al.
Fig. 4. FTIR-ATR spectra (1600–400 cm−1) of individual biopolymers, WPI, KC and WPI HIUS pre-treated- KC CC: (a) 50% A, 2 min, (b) 50% A, 4 min, (c) 100% A, 2
min, (d) 100% A, 4 min.
3.2.3. Fourier transform infrared spectroscopy (FTIR-ATR) Fig. 3
shows the infrared spectra of both, individual biopolymers WPI, KC
and WPI-KC complexes. WPI spectrum exhibited a strong
absorption band at 3270 cm−1 corresponding to hydroxyl
contraction vibrations attributed to hydrogen bonds of water.
Further, at 3065 cm−1, there was a weak absorption that
corresponds to stretching N–H vibration (amide B), the absorption
band at 2955 cm−1 corresponds to stretching vibration of –CH. It is
possible to observe on the fingerprint region (1800 to 900 cm −1)
the vibrations of the characteristic protein groups, the broad
bands in the spectrum centered at approximately 1640 cm −1 and
1520 cm−1 were associated with amide I (α-helix and β-sheet
structures of proteins) and amide II (combined (N–H) and (C–N))
respectively, contributing to the peptide bond group vibrations of
proteins. The band at approximately 1450 cm −1 resulted from the
deformation bending of C–H bonds in the > CH 2 groups and the
band at 1390 cm−1 corresponded to the stretching of C = O in the –
COO groups. The broad peak band ~1250 cm−1 corresponds to the
signal of the amide III groups [42,55,56,57].
On the other hand KC, presented its typical absorption bands
at 847 and 926 cm −1 indicating the presence of D-galactose-4
sulfate and 3,6anhydro-D galactose, molecular units of the KC
[56,57]. The vibration at 1041 cm−1 is attributed to the vibrations of
the CO and C-OH bonds [57]; also, the adsorption band at 1077
cm−1 corresponds to the CO bond of 3,6 anhydrous D-galactose
[56,57].
Meanwhile CC spectra, presented the typical bands of
absorption of WPI (1600–1700 cm −1) and KC (900–1400 cm −1). The
possible electrostatic interactions between both biopolymers may
be a visible inversion in the band that corresponds to the
vibrations of the CO bond of the 3,6 Anhydrous-D-galactose (1041
cm−1) and the stretch CN bond of the primary protein amine (1077
cm−1). There was also a modification in the band shape at 1238
cm−1 that corresponds to the vibration of the SO bond of KC sulfate
groups and the CN bond of the WPI primary amine. Other points
of possible electrostatic interaction with visible changes (3283
cm−1 and 3366 cm−1) were those that correspond to the vibration
of the NH bonds in the case of protein and OH in the case of KC
[55,58], these regions had very characteristic spectrum band
profiles for proteins and polysaccharides. In the case of
coacervates, a band of neither of the two characteristic bands was
obtained, which could indicate a possible point of interaction
[11,58].
On the other hand, the FTIR-ATR absorption spectra for CC
elaborated with HIUS-treated WPI are shown in Fig. 4. As the
control, CC spectrum presented typical absorption bands of WPI
Ultrasonics - Sonochemistry 70 (2021) 105340
(1600–1700 cm−1) and KC (900–1400 cm−1), the possible
interactions between both biopolymers remains regardless of the
HIUS treatment applied to WPI.
Although differences were observed at 1041 and 1077 cm −1,
independently of the ultrasonic treatment, there was a noticeable
change in the shape of the band that corresponds to the vibrations
of the CO bond of the 3,6 anhydrous-D-galactose and to the
stretch CN bond of the primary protein amine, respectively.
Another important change in the band shape was found at 1238
cm−1, corresponding to the asymmetric vibration of the sulfate
groups of KC and the asymmetric vibration of the CN bond of WPI
[42,55,57,59]. There were also differences regarding CC control, in
the region between 1400 and 1500 cm −1 corresponding to the
vibrations of the carboxylate groups and C-OH vibrations [60]. In
general terms, stability in the coacervate system is proportional to
the number of interactions between the KC and the WPI, i.e. more
interaction, greater stability [61]. The conformational changes
presented in HIUS WPI, influenced the way in which both
biopolymers interact electrostatically; however, the mainly groups
involved in the CC interactions remains and were NH +, CN, –OH for
WPI and, –COH, –COOH, –OH, –SO4 groups in the case of KC.
As it is well known, HIUS implosions can cause extreme
localized changes in temperature and pressure at the sites known
as hot spots; in addition to all the already mentioned physical
changes associated with cavitation, there are also selected
chemical effects including the formation of free radicals by
different sonochemical reactions that may affect aroma, taste,
mouthfeel and technological features of foodstuffs [61,62]. The
behavior of proteins under sonication mainly depends on their
structural complexity and on the type of dominant secondary
structure present (α-helix or β-sheet) [27,63]. It has been
suggested that free radicals produced by water sonolysis, such as
superoxides (H2O.) have an important role in in the modification of
protein structures inducing disulfide crosslinking of protein
molecules in aqueous medium [61]. However, in our scenario, the
impact of free radicals is expected to be very mild. Short ultrasonic
processing times (as those used in the present study) have been
observed to cause a reduced freeradical formation and less
pronounced related off flavors in food matrices [64]. Besides, the
presence of KC could have helped to reduce any free radical-
related damages in the sample. Sulphated polysaccharides
extracted from brown and red seaweeds such as KC have been
shown to inhibit the formation of free radicals and their sulphation
degree was found to be directly related to their radical scavenging
activity, with KC exhibiting mild antioxidant properties [65].
8
S.A. Vargas, et al.
Consequently, the impact of free radicals in the WPI-KC complex
coacervated systems was considered to be negligible.
Table 5 WPI, KC and CC Viscosity (25
°C).
Viscosity mPa*s (25 °C)
HIUS treatment on WPI
KC
– 57.44 ± 1.17
50%A, 2 min
50%A, 4 min
100%A, 2 min
100%A, 4 min
Different letters in the same column present significant difference (p <
0.05).
PL = Power law.
HB = Herschel-Bulkley.
3.3. Functional properties of WPI-KC complex systems
3.3.1. Rheology
The viscosity in dispersed proteins depends on different
molecular properties such as size and shape, as well as their
protein-solvent interactions, hydrodynamic volume and molecular
flexibility in the hydrated state [62]. In the present study, changes
in viscosity because of protein- polysaccharide interaction were
desirable. CC presented a different viscosity level compared to
individual biopolymers (Table 5), which represented the formation
of a new structure. It is possible to observe a significant increase
(~20 times) on CC viscosity (~20 mPa*s, 25 °C) compared to the
WPI (1.16 ± 0.07 mPa*s, 25 °C). This increase in viscosity can be
attributed to the formation of larger molecular entities due to
complex coacervation, improving the viscoelastic properties of the
system [1,12]. Nevertheless, regardless the HIUS treatment
applied, no significant changes (p < 0.05) on viscosity level of WPI
and CC were detected.
On the other hand, the rheological behavior of the coacervates
is that of a pseudo plastic fluid, i.e., when shear speed increases,
viscosity decreases. For this reason, CC viscosity was adjusted to
the mathematic models used for pseudoplastic fluids: Power law
(PL) and Herschel–Buckley (HB). According to the results, the HB
model presented lower RMSE value than PL model; based on that,
it was a better option to use as predictive equation for a
rheological behavior model.
3.3.2. Foaming capacity
Several studies have shown that ultrasonic treatment can
improve WPI foam stability [35,67], however an excessive
treatment could generate a drastic loss in the foaming ability, due
to aggregation and polymerization induced by treatments [15]. In
order to known the influence of HIUS on WPI-KC complex
coacervation, foaming capacity was evaluated.
Foaming properties of proteins are determined by its
molecular structure, HIUS could affect these properties by
inducing conformational changes in the secondary, and tertiary
structures, as well as changes in both; strong and weak bonds,
including hydrogen bonds, and also on structures that have been
demonstrated to determine the foaming properties such as,
hydrophobic interactions, electrostatic bonds and disulfide bonds
[15]. In this work, according to the results of physicochemical
characterization, through HIUS treatment, it was possible to
increase surface hydrophobicity (Table 1), to reduce particle size
(Table 2) and to induce changes on the structural conformation
(Table 3) of WPI. The influence of these effects on the foaming
capacity of WPI HIUS-KC complex coacervates, was evaluated
through the following parameters: foam stability (%), volume
Ultrasonics - Sonochemistry 70 (2021) 105340
increment or overrum (%) and foam expansion ability (%) (Table
6). HIUS treatment on WPI has a significant influence (p < 0.05),
CC adjustment, RMSE
WPI CC PL HB
a a
1.16 ± 0.07 28.09 ± 1.17 1.71 0.06
1.17 ± 0.08a 22.18 ± 2.13a 0.15 0.03
1.17 ± 0.05a 30.39 ± 0.6a 0.13 0.09
1.24 ± 0.02a 23.26 ± 4.20a 0.11 0.06
1.25 ± 0.04a 23.01 ± 2.05a 0.13 0.04
increasing foaming capacity as the amplitude and treatment time
increase, i.e., the most intense treatments (100%, 2–4 min)
produce more stable foams with a major expansion ability and
overrun. These improvements in CC foaming capacity could be
related with the partial denaturation of proteins resulting in
molecular rearrangements that can lead to form thicker rigid
interface films [41].
Although there were improvements in foaming capacity due to
HIUS, the use of KC is also a key factor in foam stability of CC,
showing a synergistic effect, WPI is able to spread more rapidly in
the air–water interface due to its high molecular flexibility, which
results in a foam brittle and characterized by large pores [31]. The
addition of KC resulted in higher foam stability, forming a thicker
and more viscous film with the ability to retain air for longer
periods of time. Protein must migrate to the air–water interface
and unfold to expose hydrophobic groups towards the gaseous
phase and hydrophilic groups to the aqueous phase to form a
viscoelastic film around the air bubble. In this context,
electrostatic complexation influences both diffusion of proteins
into the interface and re-alignment once there [40].
Polysaccharides had no affinity for the air–water interface, but
encourage protein–protein interactions, which lead to developed
a multilayer cohesive protein film on the interface that prevents
foam to collapse and allows more stable foams formation [31].
These interfacial networks also tend to show a ‘jamming effect’,
which reduces foam drainage and maintains the structural
integrity of the system [68].
Fig. 5 shows the optical microscopy structure of foams, it is
important to notice that in all the treatments applied (Fig. 5b-5f),
bubbles with spherical morphology and well-defined lamellae
were produced, it is not possible to observe empty spaces
between the air bubbles, which could contribute to enhanced
foam stability, resulting in higher foaming capacity. By contrast, on
the WPI foam the spaces between bubbles are visible (Fig. 5a)
resulting in a not stable foam compared to CC. Electrostatic
complexation is expected to influence both diffusion of proteins to
the interface and re-alignment once there [40]. The use of KC and
HIUS pretreated WPI, enhance the resistance against bubble
collapse, forming stronger lamellas and adsorbed films [69].
3.3.3. Emulsifying properties
Ultrasound treatment increases protein surface
hydrophobicity due to the mechanical effects of cavitation,
causing structural changes on αhelix and β-sheets, as a
consequence, protein unfolding results, modifying the secondary
and tertiary structures of the protein; internal hydrophobic
regions of protein are unmasked, this resulted in a loosening of
the protein folding and a consequent change in the emulsifying
capacity of the protein [36,50]. There are several studies that have
reported the influence of sonication on emulsions, forming smaller
droplets, decreasing interfacial tension, with significant increases
in surface hydrophobicity, solubility, emulsifying activity index
(EAI), and emulsifying stability index (ESI) [37].
9
S.A. Vargas, et al.
According to the results, due to HIUS treatment there was a
significant (p < 0.05) increase on EAI, ESI, and the stability of the
emulsions (%), related to the induced conformational changes and
the increase of WPI surface hydrophobicity [66] (Table 7). On the
other hand, the effectiveness of KC influencing emulsifying
properties of WPI seems to be related to the linear charge density
of the carrageenan and possibly its conformation in solution [12].
During the emulsification
Table 6
Complex coacervates foaming capacity.
Sample HIUS treatment on WPI
WPI – 73.40 ± 2.27
Complex coacervates – 71.17 ± 1.74c
50%A, 2 min
50%A, 4 min
100%A, 2 min
100% A, 4 min
Different letters in the same column present significant difference (p <
0.05).
process, protein-polysaccharide complexes migrate to the oil–
water interface and then realign to form viscoelastic films around
the oil bubbles, where the hydrophobic groups of protein were
oriented to the oil phase and the hydrophilic groups of
polysaccharide to the aqueous [40].
The percentage of emulsion stability relates the degree of
emulsion breakdown over a defined time period, after one month
the emulsion corresponding to CC: WPI treated by 100%
amplitude, 2 min remains without phase separation, being a
significant difference (p < 0.05) between this treatment and the
other applied. The increase in WPI emulsion stability when is
complexed with carrageenan may be due to charge repulsion
between droplets, viscoelastic film formation with protein–
polysaccharide complexes saturating the oil–water interface, steric
stabilization caused by the carrageenan chains and an increase in
the viscosity of the continuous phase [51].
3.3.4. Solubility determination
HIUS treatment did not presented a significant (p < 0.05)
influence on the solubility of the samples (~90%) (Table 8). The
polysaccharide seems to inhibit protein–protein interactions
resulting from a treatment that affects protein structure, in which
solubility is likely to be lost, i.e., the complexation between
proteins and sulfated polysaccharides resulted in protection
against the loss of solubility, by blocking the protein hydrophobic
sites [40,70].
3.3.5. Gelation properties
Although both biopolymers, individually, have gelling
properties, after gelling procedure, none of CC systems could form
a gel. Above 60 °C, temperature required for WPI gelation, a
separation between WPI-KC occurred (data not shown). The
physicochemical properties of proteins and polysaccharides are
influenced by temperature. Polysaccharide and protein complex
formation is mainly driven by various non-covalent interactions,
like electrostatic, H-bonding, hydrophobic, and steric interactions;
the extent of hydrogen bonding and hydrophobic interaction
depends on temperature [71]. Hydrogen bonding is a moderately
strong bond, which becomes relatively insignificant at high
temperature, these bonds are ionic in nature and refer to the
interaction between hydrogen atoms attached to an
electronegative atom (oxygen, sulfur) and other electronegative
atom (the sulfur of sulfate group), a classic example of hydrogen
bonding has been shown in the complex coacervation [2]. In this
case, complex coacervation was lost with the increase in
temperature, which is in agreement with the FTIR results (Figs. 3
Ultrasonics - Sonochemistry 70 (2021) 105340
and 4) where an electrostatic interaction between both
biopolymers was observed.
3.3.6. Application of WPI-KC complex systems based on their
functionalproperties
Whey proteins have been used as an additive to formulate
products rich in protein and / or low in lactose, also by its
improved biological value, conferred by its content in bioactive
Foam stability (FS%) Volume increment (VI%) Foam expansion ability (FE%)
c c
75.00 ± 0.11 173.75 ± 1.44c
77.50 ± 2.04c 177.5 ± 2.04c
77.85 ± 1.71b 126.56 ± 2.77b 226.56 ± 2.77b
78.00 ± 1.29b 125.63 ± 1.61b 147.81 ± 225.63 ± 1.61b 247.81 ±
90.25 ± 1.26a 2.13a 2.13a
90.0 ± 0.82a 148.75 ± 1.02a 248.75 ± 1.02a
peptides with biological properties, such as antihypertensive,
antioxidant, antimicrobial and immunomodulatory activities and
also because of its functional properties such as, gelling, foaming
and emulsification [4,72]. Based on this, it is feasible to apply
whey protein as a functional ingredient with bioactive properties;
in this sense, complex coacervates represent an important tool for
food manufacturers to enhance food quality and even nutritional
attributes [1,51]. Rheological properties as well as interfacial
properties in emulsions and foams are the prerequisite for the
successful use of protein- polysaccharide mixed systems [1].
Nowadays, in fermented dairy products, such as sour milk and
yogurt drinks, CC represent a powerful tool to modulate viscosity
and heat stability by associative interactions, also have been used
to produce meat analogs and fat substitutes or replacers.
Applications as texturing agents include protein- polysaccharide
core–shell microparticles utilized as a cream or fat substitute. CC
also have been utilized as structuring agents with the capacity to
bind and/or structure water in low-fat confectionery products and
to stabilize interfaces in products such as mayonnaises and
foamed emulsions [1]. Although proteins are efficient emulsifiers,
do not produce emulsions that are stable under broad
environmental conditions (e.g., pH, ionic strength, etc.); as
Krempel et, al. suggest, many studies have proposed using
polysaccharide-protein complexes as alternative sources for gum
arabic or modified starches in beverage emulsions [73,74].
Fundamental understanding of the interactions at the molecular
and colloidal levels will provide a strong basis to exploit the
physical functionality of such complex systems in different
applications (e.g. microencapsulation technology, imparting
specific sensory characteristics, time/temperature/pH/ionic
control-release, emulsion stability, etc. [2]. Finally, studies in
complex food systems are still needed to assess food applications;
however, the initial results offer promising behavior in several
products, such as sherbet and aerated foams obtained from acidic
dairy products [1].
4. Conclusions
Although complex coacervation has been widely described, the
use of HIUS technique helped to improve some of its functional
properties. According to FTIR-ATR spectra analysis, complex
coacervation was carried out through multiple electrostatic
interactions and hydrogen bonds, given as a result, spherical and
defined forms, that were visualized by optical microscopy.
Deconvolution analysis suggest, that WPI structure was modified
by HIUS treatments and surface hydrophobicity increased,
meanwhile OD and particle size decrease. The treatment with the
highest increase in disordered structures and β-sheets,
hydrophobicity and therefore better functional properties
10
S.A. Vargas, et al.
(foaming and emulsifying) was the treatment at amplitude of
100%, 2 min. CC produced with WPI HIUS treated could increase
the foaming capacity, the stability of emulsions, add viscosity and
maintain their solubility. Based on this, the HIUS and KC complex
coacervation is an effective tool to improve WPI functional
properties, which creates a huge gap in the successful application
of the CC system, as an ingredient of high value to the food
industry.
Ultrasonics - Sonochemistry 70 (2021) 105340
CRediT authorship contribution statement
Sara A. Vargas: Investigation, Writing - original draft. : . R.J.
Delgado-Macuil: Methodology, Formal analysis, Resources,
Writing review & editing, Supervision. H. Ruiz-Espinosa:
Conceptualization, Writing - review & editing, Methodology. M.
Rojas-López:
11
S.A. Vargas, et al. Ultrasonics - Sonochemistry 70 (2021) 105340

(a) (b)

(c) (d)

(e) (f)

Fig. 5. Optical microscopy (10x), foams morphology of: (a) WPI, (b) WPI-KC CC, WPI HIUS- KC CC: (c) 50%A, 2 min, (d) 50%A, 4 min, (e) CC (100%A, 2 min), (f) CC
(100%A, 4 min).
Table 7
Emulsifying properties of complex coacervates.
Sample HIUS treatment on WPI EAI (m2/g) (25 °C) ESI (min) ES (%)

12
S.A. Vargas, et al. Ultrasonics - Sonochemistry 70 (2021) 105340
3h 1 week 1 month

WPI – 3.65 ± 0.27f 3.19 ± 0.16c 100.0 ± 0.0a 90.0 ± 0.0b 25.0 ± 1.0e
Complex Coacervates – 14.15 ± 0.20e 19.95 ± 1.05b 100.0 ± 0.0a 100.0 ± 0.0a 76.25 ± 1.77b
50%A, 2 min 22.55 ± 0.41d 36.17 ± 0.92a 63.75 ± 1.77b 63.75 ± 1.77c 68.12 ± 0.88c
50%A, 4 min 37.76 ± 0.61c 31.55 ± 0.87a 65.00 ± 3.54b 63.75 ± 3.54c 48.75 ± 1.77d
100%A, 2 min 47.40 ± 0.54a 34.65 ± 1.5a 100.0 ± 0.0a 100.0 ± 0.0a 100.0 ± 0.0a
100% A, 4 min 40.49 ± 0.41b 36.0 ± 1.29a 100.0 ± 0.0a 88.75 ± 0.0b 80.62 ± 0.88b

Different letters in the same column present significant difference (p < 0.05).
EAI = emulsifying activity index
ESI = emulsifying stability index.
ES = emulsion stability, 100% means no phase separation.
Table 8
Complex coacervates solubility determination.
Sample HIUS treatment on WPI Solubility (%) (25 °C)
WPI – 95.5 ± 0.707a
Complex Coacervates 50%A, 2 min 50%A, 4 95.0 ± 1.41a
min 92.5 ± 3.54a
100%A, 2 min 93.5 ± 4.95a
100% A, 4 min
92.5 ± 2.12a
Different letters in the same column present significant difference (p < 0.05).
Methodology, Formal analysis. G.G. Amador-Espejo:
Conceptualization, Methodology, Writing - review & editing,
Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
Financial support for this research was provided by the
Mexican National Council for Science and Technology (through
scholarship support 577084, grant CONACyT 254412), Instituto
Politécnico Nacional (SIP). The authors kindly acknowledge
Ingredion Mexico for providing the kappa carrageenan employed
in this study.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https:// doi.org/10.1016/j.ultsonch.2020.105340. References
[1] S.L. Turgeon, S.I. Laneuville, Protein + Polysaccharide Coacervates and
Complexes. From Scientific Background to their Application as Functional
Ingredients in Food Products, First Edit, Elsevier Inc., 2009. doi:
10.1016/B978-0-12-374195-0. 00011-2.
[2] K.K.T. Goh, A. Sarkar, H. Singh, Chapter 12 - {Milk} protein–polysaccharide
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