Food Hydrocolloids 61 (2016) 903e913

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

Understanding the stability mechanisms of lentil legumin-like protein

and polysaccharide foams
M. Jarpa-Parra a, 1, Z. Tian a, Feral Temelli a, Hongbo Zeng b, L. Chen a, *
a
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, T6G 2P5, Canada
b
Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, T6G 1H9, Canada

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

Article history: Foaming properties of lentil legumin-like protein were investigated in the presence of guar gum, xanthan
Received 5 January 2016 gum and pectin at different environmental pH conditions (3.0, 5.0, and 7.0). The protein foaming capacity
Received in revised form was not significantly impacted by adding polysaccharides, whereas foam stability was greatly enhanced
6 July 2016
at pH 3.0 and 5.0, leading to formation of long-life foams in most samples with the highest mean life
Accepted 11 July 2016
Available online 13 July 2016
value of 275 min at pH 5.0 in the presence of pectin. Investigation of the stability mechanisms revealed
that at pH 3.0, the presence of the coacervates stabilized the foams against collapse due to the formation
of an electrostatically cross-linked gel-like interfacial network. At pH 5.0, aggregates were formed that
Keywords:
Lentil protein
adsorbed to the interface to form stiff and thick interfacial network, avoiding foam coarsening. Aggre-
Polysaccharide gates also plugged the junctions of the Plateau borders, slowing down the drainage by a jamming effect,
Foam stability and dramatically increased apparent viscosity of the foams, thus favoring the immobilization of the
Interfacial behavior lamellar water surrounding the gas bubbles. The thermodynamic incompatibility at pH 7.0 resulted in a
Coacervation phase separation of protein and polysaccharide in the interfacial protein membrane. This induced a
Phase separation disruption of the protein layer around the bubbles making it weaker and easier to break, leading to
reduced foaming stability. The findings revealed that guar, xanthan, and pectin can improve the stability
of lentil legumin-like protein foams at mild acidic pH, creating long-life foams, which would be
particularly useful in the food industry where aerated structures must be preserved for a long period of
time before solidifying or gelling.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction interface. The presence of proteins empowers these properties due

A foam can decay smoothly or collapse very fast. Foam mean life
value is considered as an index of foam stability, which is the in-
verse of the decay constant of a foam system (Hackbarth, 2006).
Long-life foams, which normally show mean life value of 50 min or
higher (Piazza, Gigli, & Bulbarello, 2008) are particularly useful in
the food industry when products must be processed for long pe-
riods and it is important to keep the aerated structure before so-
lidifying or gelling (e.g. mousse and ice cream). Formation and
stabilization of foams are to a large extent determined by the
properties of the compounds they contain at the air/water
* Corresponding author.
E-mail address: lingyun.chen@ualberta.ca (L. Chen).
1
Current address: Graduate School of Universidad Adventista de Chile, Casilla 7-
D, Chill
an, Chile.
to a combination of their surface activity, and electrostatic and
steric mechanisms (Foegeding, Luck, & Davis, 2006).
Lentil is a leguminous plant low in fat and high in dietary fibre
and other nutrients, such as protein, dietary fiber, vitamins and
minerals (Roy, Boye, & Simpson, 2010; Thavarajah, Thavarajah, &
Sarker, 2009) and its consumption has been associated with
several health benefits (Barbana & Boye, 2011; Roy et al., 2010).
Lentil contains 20.6e31.4% protein with legumin-like protein
(~50%) as the major globulin fraction (Urbano, Porres, Frias, & Vidal-
Valverde, 2007). Generally, it is accepted that legumin is a hexamer
with a molecular weight (Mw) of about 320e380 kDa, which con-
sists of six polypeptide pairs that interact non-covalently. Each of
these polypeptide pairs is comprised of an acidic subunit of about
40 kDa and a basic subunit of about 20 kDa, linked by a single di-
sulfide bond (Urbano et al., 2007). Lentil legumin-like protein has
an isoelectric point (pI) around 4.6 with balanced hydrophilic
(~38%) and hydrophobic (~40%) residues. It also possesses both high

http://dx.doi.org/10.1016/j.foodhyd.2016.07.017
0268-005X/© 2016 Elsevier Ltd. All rights reserved.
904 M. Jarpa-Parra et al. / Food Hydrocolloids 61 (2016) 903e913

surface hydrophobicity and solubility when pH is deviated from its 2. Materials and methods
pI (Jarpa-Parra et al., 2015). Thus, lentil legumin-like protein
demonstrated excellent foaming capacity and stability at pH 5.0 2.1. Raw materials
and 7.0, respectively (Jarpa-Parra et al., 2015).
Protein foam properties can be improved by the addition of Lentil legumin-like protein extract (83% w/w) was obtained by
polysaccharides, and the foam stability depends on the interactions rate-zonal centrifugation using a sucrose lineal density gradient as
between these two kinds of biopolymers and the ability of their described in our previous work (Jarpa-Parra et al., 2015). The
mixture to retard gravity-induced drainage by controlling the remaining part consisted of 5.0% of water, 2.4% of ash, 1.6 of lipids,
rheology and network structure of the continuous phase (Narchi, and 8.0% of carbohydrates (mainly starch). Guar and xanthan gums,
Vial, & Djelveh, 2009; Rodríguez-Patino & Pilosof, 2011). Complex Rhodamine B, and Calcofluor White (Fluorescent brightener 28)
coacervation, miscibility, and segregation are the possible phe- were obtained from Sigma-Aldrich Canada Co. (Oakville, Ontario,
nomena that arise in aqueous solutions of protein and poly- ON, Canada). The LM Pectin was provided by MP Biomedicals, LLC
saccharide mixtures (Narchi et al., 2009). Using anionic (Solon, OH, USA). All other chemicals were reagent-grade.
polysaccharide as an example, if the pH of the aqueous medium is
reduced to below the isoelectric point (pI) of the protein, complex
coacervation occurs as a result of net electrostatic attraction be- 2.2. Preparation of the protein-polysaccharide mixtures
tween the oppositely charged biopolymers coupled with phase
separation, one rich in the complexed biopolymers and the other Pectin, guar or xanthan gum (1 mg/mL) solution was mixed with
phase depleted in both. Above the isoelectric point, because of the the lentil legumin-like protein (10 mg/mL) solution by magnetic
repulsive electrostatic interactions and different affinities towards stirring. Mixtures were prepared at three different pH levels (3.0,
the solvent, thermodynamic incompatibility of protein and poly- 5.0, and 7.0) adjusted using 0.1 M HCl or NaOH.
saccharide occurs. Above a critical concentration and/or at high
ionic strength, protein and polysaccharide segregate into different
phases. However, if their concentration is sufficiently low, they 2.3. Electrophoretic mobility
could co-exist in a single phase (miscibility) in which they mutually
exclude one another (Rodríguez-Patino & Pilosof, 2011). The electrophoretic mobility of the protein-polysaccharide
The impact of protein-polysaccharide interactions on the dy- mixtures within the pH range of 2.0e11.0 (adjusted using 0.1 M
namics of protein adsorption at the interface and the effect on foam HCl or NaOH) at 22  C was measured by laser Doppler velocimetry
capacity has been studied previously (Miquelim, Lannes, & using a Zetasizer NanoS (model ZEN1600, Malvern Instruments
Mezzenga, 2010; Sadahira et al., 2015; Van den Berg, Jara, & Ltd., Malvern, UK) as described previously (Jarpa-Parra et al., 2015).
Pilosof, 2015). Most research studied the influence of the protein-
polysaccharide interactions just at one pH value, normally below
pI. In one of the most recent studies, Ruíz-Henestrosa, Carrera-
2.4. Foam properties
nchez, and Patino. (2008) analyzed the effect of sucrose on dy-
Sa
namic surface pressure, surface dilatational properties and foam
Foaming capacity was determined as in our previous study
characteristics of soy globulins (7S and 11S) at pH 7.0 and 5.0. As pH
(Jarpa-Parra et al., 2014). Briefly, 30 mL of protein-polysaccharide
plays an important role to determine protein polysaccharide in-
mixtures were mixed for 2 min with a homogenizer (PowerGen
teractions, its impact on surface properties of the complex systems
1000, Fisher Scientific, Fairlawn, NJ, USA) at speed six. The foaming
should be investigated in a broad range of pH conditions applied for
capacity (FC) was calculated as: FC (%) ¼ (Vf 1  Vf 0)/Vf 0
100,
food applications. In addition, a fundamental understanding of the
where Vf 0 and Vf 1 represent the volumes of the protein-
impact of polysaccharide addition on the molecular structure of
polysaccharide mixture and the formed foams after homogeniza-
lentil protein at the foam interface, subsequently the foaming
tion, respectively.
properties of the complex system has never been reported.
As an index of foam stability (FS), a “mean life” t value was
The purpose of the present study was to investigate how the
determined according to Equation (1) (Hackbarth, 2006). The larger
presence of different polysaccharides may impact the lentil
the t value, the longer is the foam stand time.
legumin-like protein capacity to stabilize foams. It is hypothesized
that a synergistic effect could be achieved by modulating protein-
HðtÞ ¼ Hð0Þelt (1)
polysaccharide interactions for improved foam stability. The poly-
saccharides selected for this study are guar gum, xanthan gum, and
where H(0) is the initial foam height at time t ¼ 0, and l is the decay
pectin, representing non-ionic and anionic polysaccharides with
constant, which is a measure of foam decay. This exponential
different molecular weights. These are widely used in food appli-
relationship can be converted to a linear equation by taking the
cations as thickening or gelling agents and stabilizers because of
natural logarithm of the foam height, ln(H(t)), vs time, where the
their water-binding properties. Guar gum is a non-ionic gal-
slope value corresponds to el. t is the inverse of a decay constant of
actomannan with Mw of 106 Da. Xanthan gum is a stiff high mo-
a foam system.
lecular weight anionic polysaccharide with Mw of 2
106 to
5
107 Da that can form highly viscous solutions. Pectin is a ln½HðtÞ ¼ ln½Hð0Þ  lt (2)
commonly used anionic polysaccharide with Mw of 2
104 to
4
105 Da. The two major commercially available pectins have
d½lnðHÞ 1
typical DE values of 70% and 35%, corresponding to high methoxyl ¼ l ¼ (3)
(HM) and low methoxyl (LM) pectin, respectively (De Jong & Van de dt t
Velde, 2007). The impact of polysaccharides of different molecular Foam drainage was also calculated by measuring the volume of
structures (i.e. molecular weight and surface charge) on protein the drained liquid from the foams in a graduated cylinder over a
surface properties (surface tension, dilatational and shear period of 1 h and expressed as the liquid fraction (ε) in the foam
rheology), and subsequently foaming functionality was systemati- ((volume of liquid  volume of drained liquid)/volume of foam)
cally investigated at different environmental pH values. (Salonen, In, Emile, & Saint-James, 2010).
 M. Jarpa-Parra et al. / Food Hydrocolloids 61 (2016) 903e913
2.5. Surface properties
2.5.1. Surface tension of protein-polysaccharide solutions
The surface tension of the protein-polysaccharide mixtures was
measured at 22  C with a DHR3 rheometer (AT Instruments-Waters
LLC, New Castle, DE, USA) using a Platinum/Iridium Du Noüy ring
(diameter of 10 mm). The ring was immersed in the mixture so-
lution and gradually raised above the surface. The maximum value
of the force at the detachment of the ring from the surface of the
solution was recorded for surface tension calculation (Cases,
Rampini, & Cayot, 2005).
2.5.2. Shear rheology measurement of protein-polysaccharides
foams
The rheological studies were conducted according to Ptaszek
(2013). Preliminary trials were conducted for selection of the
suitable geometry to reduce slip of the foam on the sensor's sur-
faces and determination of the appropriate gap size to prevent
crushing and destruction of gas bubbles. The geometry of the
sensor and the size of the measurement gap were empirically
selected using 95% reproducibility of the results as a criterion. Then,
the viscoelastic properties of the protein-polysaccharide foams
were measured as a function of time. Coalescence of droplets was
not considered when the rheology measurements were done.
Samples were analyzed by means of a DHR3 rheometer (AT
Instruments-Waters LLC, New Castle, DE, USA) fitted with a cone
and plate attachment with a diameter of 35 mm and angle of 2 .
Strain sweep tests with strain amplitudes ranging from 0.01% to
100% were performed at 0.6283 rad/s in order to establish the linear
viscoelastic range. Based on these results, frequency sweep mea-
surements (0.1e100 rad/s) were carried out at a strain amplitude of
1%; a value smaller than the critical value for linear viscoelasticity.
Oscillatory measurements were performed in the linear region at a
frequency of 0.6283 rad/s and strain of 1%, and the storage modulus
G0 , loss modulus G00 , and tan (d) ¼ G00 /G0 were recorded. In addition,
shear rate-shear stress and shear rate-apparent viscosity data were
collected as shear rate was increased linearly between 1 and
100 s1 over a total run time of 5 min. During the analysis, the
sample was kept at 22  C.
2.6. Protein conformation in foams with polysaccharide
Pectin, guar, xanthan gum (1 mg/mL) and lentil legumin-like
protein (10 mg/mL) were dissolved in D2O by magnetic stirring.
Mixtures were prepared at three different pH levels (3.0, 5.0, and
7.0) adjusted using either NaOD or DCl (0.1% v/v). To ensure com-
plete H/D exchange, samples were prepared 2 days before and kept
at 4  C prior measurement. Infrared spectra of foams were recorded
at 22  C using a Nicolet 6700 spectrometer (Thermo Scientific,
Madison, WI, USA) and Fourier self-deconvolutions were per-
formed using the software provided with the spectrometer to study
the amide I region of the protein. Band narrowing was achieved
with a full width at half maximum of 10 cm1 and with a resolution
enhancement factor of 1.0 cm1. Protein in solution before foaming
formation and in the drained liquid coming down from the foams
were also analyzed for comparison.
2.7. Microstructure of protein-polysaccharide mixtures and their
foams
Images of the protein-polysaccharide mixtures were taken with
a Zeiss Axio Imager M1 fluorescence microscope with a
10
objective (Carl Zeiss Microscopy, Jena, Germany) using the
filter set 10 (excitation: 450e490 nm, emission: 515e565 nm).
Rhodamine B was used for labeling of lentil legumin-like protein
905
and calcofluor white was used for labeling of the polysaccharides.
After adding the fluorescence dye, the protein and the poly-
saccharides were separately stirred for 12 h at 22  C, dialyzed
against distilled water in the dark, and freeze-dried. Protein-poly-
saccharide suspensions were prepared in darkness and at the same
concentration as described previously in section 2.2.
The microscopic structure of the protein-polysaccharide foams
was examined using a confocal laser scanning microscope (CLSM)
Zeiss LSM710 (Carl Zeiss Microscopy, Jena, Germany) with a
20
objective. Foams were prepared immediately before mea-
surement. Protein-polysaccharide mixtures utilized to prepare the
foams were dyed as indicated for fluorescence microscopy. Foam
samples were loaded on the microscope slides and the fluorescent
images were analyzed at the wavelengths of 420 and 516 nm for
Calcofluor and Rhodamine B, respectively. Images were processed
with the ZEN 2009 LE software (Carl Zeiss AG, Oberkochen,
Germany).
2.8. Statistical analysis
All samples were prepared and tested in triplicate and the re-
sults are presented as mean ± standard deviation. One-way or two-
way analysis of variance (ANOVA) was carried out using Origin 9.1
software (Origin Lab Corporation, Northampton, MA, USA), and
statistical differences among sample means were determined using
Tukey's test at 95% confidence level.
3. Results and discussion
3.1. Fluoresce microscopy observation
Fluorescence microscopy was used to observe interactions in
protein-polysaccharide systems. Fluorescent micrographs of the
mixtures at pH 3.0 are shown in Fig. 1A, D, and G with protein
visualized as red and polysaccharide as green/yellow. In the guar
gum-protein solution, small polysaccharide particles were homo-
geneously distributed in the protein matrix, indicating co-solubility
between the polymers. Yet, some larger protein-polysaccharide
complex structures were observed, suggesting a primary coacer-
vation of the system (Zhang, Zhang, & Vardhanabhuti, 2014).
Although guar gum is considered to be a non-ionic polysaccharide,
it exhibits a small negative charge (0.40 mV) at pH 3.0. Therefore,
opposite charges between lentil legumin-like protein and guar gum
could allow formation of weak electrostatic attraction. This kind of
complexation between a non-ionic polysaccharide and a protein at
pH 3.0 was previously observed between egg white and hydrox-
ypropylmethylcellulose (HPMC) (Van den Berg et al., 2015).
Furthermore, complexes may be formed by hydrophobic in-
teractions between guar gum and lentil legumin-like protein which
shows a strong surface hydrophobicity index at this pH (Jarpa-Parra
et al., 2015), as previously observed in whey protein and guar gum
mixtures, and egg white and HPMC mixtures (Van den Berg et al.,
2015; Zhang et al., 2014). At pH 3.0 (below pI), coacervates are
normally formed between protein and anionic polysaccharide such
as xanthan and pectin due to the attractive interactions between
the anionic groups on the polysaccharide chains (-COO-) and the
cationic groups on the protein chains (-NHþ 3 ) (Ganzevles,
Zinoviadou, Van Vliet, Stuart, & Jongh, 2006; Qiu, Zhao, &
McClements, 2015). The mixture of lentil legumin-like protein
and pectin (Fig. 1 G) shows two main domains: one composed of
lentil legumin-like protein and another one with protein-pectin
complex, both remaining loosely compacted. This phase structure
could represent the macromolecular complex stage during coac-
ervate formation (Sadahira et al., 2015). However, in the protein-
xanthan gum mixture, the micrograph (Fig. 1 D) shows well
906 M. Jarpa-Parra et al. / Food Hydrocolloids 61 (2016) 903e913
Fig. 1. Fluorescent micrographs of lentil legumin-like protein mixed with guar gum (A, B, C), xanthan gum (D, E, F), and pectin (G, H, I) at pH 3.0 (first column), pH 5.0(second
column), and pH 7.0 (third column).
defined complexes, clearly separated from the solvent phase. The
structure difference of the above two mixtures is due to the higher
charge of xanthan gum than pectin. It is expected that when a
highly negatively charged polysaccharide is mixed with the posi-
tively charged protein, they will show strong attraction to form
polyelectrolyte complexes, which can further lead to phase sepa-
ration (Sadahira et al., 2015). The structure of the protein-
polysaccharide complex is also related to the stiffness of the poly-
saccharides. The more rigid the polysaccharide chain, the denser
are the coacervates that will be formed with well-defined struc-
tures (Turgeon, Schmitt, & Sanchez, 2007). Xanthan gum has the
highest rigidity, followed by pectin and guar gum (Turgeon et al.,
2007; Zhang et al., 2014). Previous studies on b-lactoglobulin in
the presence of polysaccharides (Turgeon et al., 2007; Zhang et al.,
2014) showed that the complexes with xanthan and pectin were
denser and more compact in structure than guar gum, where the
latter remained mostly in a co-soluble state.
By adjusting the solution pH to near the isoelectric point (pI),
protein chains tend to aggregate because of the minimized inter-
molecular repulsion. In the guar gum-protein mixture at pH 5.0
(Fig. 1B), the polysaccharide was entrapped by the aggregated
protein, while in the pectin-protein mixture (Fig. 1H) both poly-
mers tended to aggregate together. Generally, the protein and guar
gum are hard to form complex at such pH, due to their low surface
charge and weak intermolecular attraction (Ghosh &
Bandyopadhyay, 2012). Electrostatic interactions could exist be-
tween lentil legumin-like protein and pectin due to positively
charged “patches” on the protein surface at pH 5.0, leading to
combined aggregation (Turgeon et al., 2007). Pectin was not
entrapped in protein aggregates probably due to its high molecular
weight and stiffness of the molecule chain. The protein-xanthan
mixture presented a different morphology (Fig. 1E). The presence
of the highly charged xanthan gum (63.67 mV) chains induced
significant electrostatic repulsive forces between adjacent poly-
saccharide chains to restrict both the size and number of protein-
protein aggregates. As a result, complexes were formed between
small protein-protein aggregates and xanthan gum molecules,
similar to those observed in lentil protein-k-carrageenan mixtures
(Aryee & Nickerson, 2014). Such aggregates broke apart into protein
and polysaccharide domains after 4h, leading to segregative phase
separation.
The mixtures of proteins and polysaccharides at pH 7.0 are
governed by segregative phenomena due to the electrostatic
repulsive interactions and different solvent affinities (Pe rez,
Carrara, Carrera-Sa nchez, Santiago, & Rodríguez-Patino, 2009;
Tolstoguzov, 1997). If the degree of incompatibility is greater,
polymer separation will occur (Doublier, Garnier, Renarda, &
Sanchez, 2000; Tolstoguzov, 1997), forming a water-in-water
emulsion system with “droplets”, rich in polysaccharide, sur-
rounded by a continuous phase rich in protein, or vice versa,
depending on the ratio of the two biopolymers (Gaaloul, Turgeon, &
Corredig, 2010). In Fig. 1C, F, and I, the protein-polysaccharide
systems at pH 7.0 clearly showed the phase separation process
with well-defined protein/polysaccharide domains, the dispersed
phase corresponding to the polysaccharide and the continuous
phase to the protein. For pectin, the phase separation with well-
defined protein and polysaccharide domains was observed after
4 h. For the mixture with xanthan gum, an “emulsion-like” struc-
ture of spherical aggregates of polysaccharides in a protein-rich
continuous phase was formed, almost immediately after mixing
(Fig. 1F) (Hemar, Tamehana, Munro, & Singh, 2001). The differences
in the phase separation behavior are likely due to the differences in
 M. Jarpa-Parra et al. / Food Hydrocolloids 61 (2016) 903e913
the charge density of the polysaccharides (De Jong & Van de Velde,
2007). In Fig. 1C, guar gum separation from lentil legumin-like
protein was not complete and protein-polysaccharide complexes
still remained.
Fig. 2. Apparent viscosity of lentil legumin-like protein-polysaccharide foams as a
function of shear rate at (A) pH 3.0, (B) pH 5.0, and (C) pH 7.0 (lentil protein: diamonds,
with guar gum: squares, with xanthan gum: circles, with pectin: triangles).
907
3.2. Surface and viscoelastic properties
The addition of the polysaccharide to protein solutions did not
significantly impact the surface tension of the mixtures (data not
shown). As shown in Fig. 2A, B, and C, the apparent viscosity (happ)
of all foams stabilized with either protein alone or protein-
polysaccharide mixtures exhibited shear thinning behavior, which
has been also observed in other protein-based foams such as milk
foams and egg white foams with or without polysaccharides
(Indrawati & Narsimhan, 2008; Jimenez-Junca, Gumy, She, & Nir-
anjan, 2011; Ptaszek, 2013). Shear thinning phenomenon in foam is
mainly caused by the disaggregation of aggregated droplets during
shearing. Also the disruption of the foam structure either by the
alignment of the protein chains in the direction of the shear force,
or by mechanical damage during the measurement (Radva nyi et al.,
2012) might be responsible in lesser extent, if the protein chains are
long or the shear rate is high.
Viscosity of a protein-polysaccharide system is expected to in-
crease as association of the polymers produces entities of larger
sizes (Ghosh & Bandyopadhyay, 2012). All foams at pH 3.0 and 5.0
exhibited a higher apparent viscosity than those at pH 7.0 at any
shear rate due to the formation of coacervates and aggregates,
respectively. A slightly higher apparent viscosity was observed at
pH 5.0 than 3.0 probably due to the larger size of the aggregates
formed near the protein pI. In general, the addition of guar gum,
xanthan gum, and pectin to the protein produced higher values of
happ than protein alone at low shear rates at both pH 3.0 and 5.0
(Fig. 2A and B). Viscosity at low shear rate can be related to long-
term stability in foams because typical shear rates experienced by
materials under gravity-induced drainage vary from 0.01/s to 1/s
(Chavez-Montes, Choplin, & Schaer, 2007; Davis & Foegeding,
2004). On the other hand, the addition of polysaccharides
decreased the foam happ value at lower shear rates at pH 7.0
(Fig. 2C), which might be related to the observed segregation
phenomenon at this pH value (Fig. 1). Indeed, at this pH, the happ
value of protein-polysaccharide foams was lower at any shear rate
than that of the protein foam alone.
Viscoelastic moduli can help to understand some of the under-
lying phenomena of foam stability as storage modulus (G0 ) is
dependent on bubble diameter (D) and liquid fraction in the foam
(ε) (Guillermic, Salonen, Emile, & Saint-Jalmes, 2009):
 
G0 f g=D gðεÞ (4)
where g(ε) is a decreasing function of only ε. Liquid fraction in the
foams of lentil legumin-like protein-polysaccharide complexes as a
function of time was also determined. During drainage, if the
bubbles get bigger, faster drainage will occur, implying a drier foam
and then even faster coarsening. Thus, G0 will continuously
decrease as the foams coarsen and collapse (Guillermic et al., 2009).
Fig. 3A, B, and C show the aging behavior of the foams repre-
sented by the storage modulus (G0 ) as a function of time. Con-
cerning the loss modulus G00 , similar qualitative features were
observed, thus results were not shown. At pH 3.0 and 5.0 (Fig. 3A
and B), all lentil legumin-like protein-polysaccharide foams
showed initial G0 values higher than that of the protein foam, while
towards the end, some showed similar value as that of protein
alone. At pH 7.0 (Fig. 3C), only the foam containing xanthan gum
had a higher G0 at t ¼ 0 compared to that of the protein, but the G'
decreased rapidly and showed a lower value than that of the foam
stabilized by protein alone after 15 min. The drainage can impact
the shear rheology measurement (Guillermic et al., 2009), which
should be investigated in the future to better clarify the foam bulk
rheological properties.
908 M. Jarpa-Parra et al. / Food Hydrocolloids 61 (2016) 903e913
Fig. 3. Storage modulus of lentil legumin-like protein-polysaccharide foams as a
function of time at (A) pH 3.0, (B) pH 5.0, and (C) pH 7.0 (lentil protein: diamonds, with
guar gum: squares, with xanthan gum: circles, with pectin: triangles).
Interaction between droplets and/or protein aggregates will
impact the shear modulus. A higher storage modulus might be the
result of the protein-polysaccharide complexes that will intensify
jamming of liquid confined to a narrow Plateau border (liquid-
carrying channels formed where three lamellae meet) between
droplets(Ptaszek, 2013). Under this jamming effect, the foam will
change from exhibiting a liquid-like behavior towards a solid-like
behavior and an increase of the G0 value might be observed
(Guillermic et al., 2009). The fluid jamming is not permanent. An
unjamming effect of the fluid occurs as a consequence of the foam
coarsening (Guillermic et al., 2009). This jamming effect may be
responsible for the continuous increase of G’ that was observed in
the foams containing guar and pectin at pH 5.0 (Fig. 3B) during the
first 10 min. This phenomenon can be related to the formation of
large aggregates (Fig. 1B and H) that were confined to the Plateau
borders in the lamellae networks, slowing down the drainage of the
liquid fraction (Fig. 4B), by reducing the Plateau border cross sec-
tion (Guillermic et al., 2009; Salonen et al., 2010). As the drainage
occurs, the amount of liquid between the bubbles changes, which
impacts the rate of diffusion of gas from bubble to bubble. Also, as
the foam coarsens, bubbles grow in size and the drainage rate in-
creases (Cha vez-Montes et al., 2007). Thus, as time passed (after
around 15 min) coarsening due to disproportionation relaxed the
confinement and fluid jamming, then yielding occurred, because
the films do not block gas diffusion driven by the difference in
Laplace pressure (Guillermic et al., 2009; Salonen et al., 2010).
The initial values of liquid fractions of protein-polysaccharide
foams at pH 3.0 (Fig. 4A) are higher than those at other pH
values. A possible explanation could be the formation of an elec-
trostatically cross-linked gel-like interfacial structure promoted by
the presence of coacervates that acted against coalescence. Such a
gel-like layer at the interface inhibited foam drainage by holding
the water in its structure (Monteux, Fuller, & Bergeron, 2004). The
gel-like structure at the interface that stabilized the foams was
previously observed in the b-lactoglobulin-pectin and the
polyelectrolyte-surfactant systems (Ganzevles et al., 2006;
Monteux et al., 2004).
At pH 7.0, the initial liquid fraction was held for a longer time
(~90 s) than at other pHs (Fig. 4). Previous studies on segregative
phase separation at the interfaces have demonstrated that phase
separation could occur if there is an effective repulsive interaction
between the polymers. Also preferential interaction of one of the
polymers with water can happen during phase separation because
of direct thermodynamic incompatibility between the components
(Semenove & Dickinson, 2010), which is likely for the poly-
saccharide used in this study and thus, leading to slow drainage
during the first stage of foam life. Also, the lack of interactions
between the phase-separated regions might explain the low G’
(Fig. 3C). There are other studies (Baeza, Carrera-Sanchez, Pilosof, &
Rodríguez-Patino, 2005; Pe rez et al., 2009) demonstrating the
improvement of foam stability under thermodynamic in-
compatibility conditions in the presence of polysaccharides. Such
studies generally attribute this phenomenon to a “concentration
effect” on proteins at the interface produced by the phase separa-
tion (Perez et al., 2009). On the contrary, in this study and some
other studies (Martínez, Baeza, Millan, & Pilosof, 2005) under
similar conditions, foam stability was reduced in the presence of
polysaccharides at pH 7.0. This difference is probably due to
different biopolymer structures and interactions.
3.3. Foaming properties
Destabilization of protein foams occurs due to simultaneous
drainage, bubble coalescence, and disproportionation (Murray,
2007). Polysaccharide addition might impact stability depending
on their interactions with protein, both at the interface vicinity and
in the bulk, which are also influenced by the polysaccharide
chemical structure, bulk viscosity, and their concentration in so-
lution (Pe rez, Carrera-Sanchez, Rodríguez-Patino, Rubiolo, &
Santiago, 2012). All the samples demonstrated good foaming ca-
pacity (FC) as shown in Fig. 5A with FC values in the range of
358e478%. Under all the pH levels tested, the FC values of the
 M. Jarpa-Parra et al. / Food Hydrocolloids 61 (2016) 903e913
Fig. 4. Liquid fraction in the foams of lentil legumin-like protein-polysaccharide as a
function of time at (A) pH 3.0, (B) pH 5.0, and (C) pH 7.0 (lentil protein: diamonds, with
guar gum: squares, with xanthan gum: circles, with pectin: triangles).
mixtures were close to those measured for lentil legumin-like
protein alone, suggesting that the selected polysaccharides did
not further improve the foaming capacity of the lentil legumin-like
protein. However, the polysaccharides had a significant effect on
the foam stability (FS), depending on the pH as shown in Fig. 5B. At
pH 3.0, guar gum, xanthan gum, and pectin increased the mean life
909
Fig. 5. (A) Foaming capacity and (B) foaming stability of lentil legumin-like protein-
polysaccharides mixtures as a function of pH. (black bar: lentil legumin-like protein,
dark gray bar: with guar gum, gray bar: with xanthan gum, light gray bar: with pectin).
Bars without a common letter have a significant difference (p < 0.05). a,b,c: Significant
difference between samples at fixed pH. x,y,z: significant differences between pH value
for the same sample.
of lentil legumin-like protein foam from 26 to 29, 87, and 42 min,
respectively. At pH 5.0, a strong synergy in the foam stability
occurred and the stability of the foams dramatically increased by
the presence of polysaccharides with the mean life value increasing
from 51 min to 126, 117, and 275 min, with guar, xanthan, and
pectin, respectively (Fig. 5B). At pH 7.0, none of the polysaccharides
improved the foam stability, instead a significant decrease FS value
was observed compared to the control sample (Fig. 5B). The good
foaming stability is particularly useful in the food industry where
foam-based products must be processed for long periods, and long-
life foams are capable of keeping the aerated structure before so-
lidifying or gelling (e.g. mousse and ice cream).
The presence of coacervates at pH 3.0 could stabilize foam in-
terfaces against disproportionation as a result of the formation of
electrostatically cross-linked interfacial networks (Miquelim et al.,
2010; Rullier, Axelos, Langevin, & Novales, 2009). Also, the small
coacervates could act as Pickering particles that are able to stabilize
foams by the formation of a coherent structural barrier of adsorbed
particles at the surface of dispersed bubbles (Dickinson, 2015;
Kuropatwa, Tolkach, & Kulozik, 2009). In addition, bubble coales-
cence is hindered due to electrostatic or steric repulsions between
910 M. Jarpa-Parra et al. / Food Hydrocolloids 61 (2016) 903e913

the coacervate stabilized air bubbles (Rullier et al., 2009). Because

of its higher charge density and more rigid structure, xanthan gum
might produce denser complexes that would build a thicker
interfacial layer (Ganzevles, Kosters, Van Vliet, Cohen Stuart, & de
Jongh, 2007), hence a more stable foam.
At pH 5.0, the strong synergistic effect could be related to the
adsorption of lentil legumin-like protein-polysaccharide aggre-
gates at the interfacial layer to form stiffer networks and their effect
on the viscoelastic behavior of the interfacial layer and the bulk
foam (Wan, Wang, Wang, Yuan, & Yang, 2014). Turgeon et al. (2007)
reported that protein-polysaccharide aggregates increased the
viscosity of the interfacial layer and they could form strong, thick,
and dense viscoelastic networks at the interface that exhibited low
gas permeability and improved foam stabilization properties. These
aggregates might also be able to plug the junctions of the Plateau
borders, so that the drainage of the liquid would slow down, further
contributing to foam stabilization (Lexis & Willenbacher, 2014). The
jamming effect significantly reduced the foam drainage, subse-
quently further contributed to the excellent foam stability at pH 5.0
(Ptaszek, 2013), especially for the lentil legumin-like protein-pectin
foam. Additionally, in this foam, the low net surface charge of the
aggregates regulated by the charge density of the pectin, may allow
stronger cohesion between aggregates within the adsorbed layer,
which, along with their size could have contributed to building of a
thicker interfacial layer (Ganzevles et al., 2007). Hence, greatly
improved foam stability was observed with the highest FS value.
The jamming effect was not observed at pH 3.0 where coacervates
existed (Fig. 3A) likely due to the smaller size of the complexes
observed at this pH value (Panouille , Durand, & Nicolai, 2005).
Additionally, the foam viscosity correlated well with the sta-
bility of the foams. The higher viscosity at lower shear rate of lentil
legumin-like protein-polysaccharides at pH 3.0 and 5.0 could favor
the immobilization of the lamellar water surrounding the gas
bubbles, improving the stability of the foam against drainage and
coalescence (Baeza et al., 2005). This phenomenon was also
observed for egg white-pectin coacervates at acidic pH when the
protein:polysaccharide mixing ratio was >1 (Sadahira, Rezende
Lopes, Rodrígues, & Netto, 2014). At this mixing ratio, the elec-
trical neutrality was not reached, leading to the formation of both
soluble complexes and non-complexed protein. The presence of
soluble complexes in the lamella increased the viscosity while the
non-complexed protein was responsible for film formation around
the bubbles. Then, these proteins act as anchor for the complexes to
build a secondary layer, which contributed to the formation of a
more stable film, inhibiting the bubble coalescence (Baeza et al.,
2005; Sadahira et al., 2014). Also, the soluble complexes may in-
crease the viscosity by forming gel-like structures, providing some
rigid and thick bridges between the bubbles, and thus the liquid
drainage was slowed down when compared to the foam formed
only with the protein (Sadahira et al., 2014). A similar explanation
can be applied to the lentil legumin-like protein systems in the
presence of polysaccharides in this study.
Molecular structure and morphology of the interface were
investigated to further understand how these characteristics
impacted the foam properties.

3.4. Foam structure

3.4.1. FTIR spectra of protein at the foam surface

Our previous study (Jarpa-Parra et al., 2015) revealed that at pH
3.0, the adsorbed lentil legumin-like protein was characterized by
unordered structure (1642 cm1), b-turns (1661 and 1677 cm1),
and some aggregates (1624 cm1). At pH 5.0, a strong aggregation
was observed (1620 cm1), as well as b-sheets (1634 cm1) and b-
turns (1663 and 1677 cm1); whereas at pH 7.0, a major peak at
Fig. 6. Fourier-deconvoluted FTIR spectra of lentil legumin-like protein-polysaccharide
foams at the foam surface. (A) pH 3.0, (B) pH 5.0, and (C) pH 7.0 (lentil protein: black,
with guar gum: red, with xanthan gum: blue, with pectin: green). (For interpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
 M. Jarpa-Parra et al. / Food Hydrocolloids 61 (2016) 903e913 911

around 1651 cm1 corresponding to a-helix could be identified,
along with b-sheets (1634 and 1683 cm1) and b-turns
(1670 cm1).
The Fourier-deconvoluted FTIR spectra of lentil legumin-like
proteins adsorbed at the foam surface in the presence of poly-
saccharides are shown in Fig. 6. Protein in the drained liquid of the
foam showed the same spectra as that in protein solution without
foam formation. However, a notorious difference was found for the
protein in the foam. Therefore, it was reasonable to believe that the
observed change of protein conformation was due to the adsorp-
tion of the protein at the interface. Based on these spectra, it is
suggested that lentil legumin-like protein underwent different
conformational changes at the foam interface, in the presence of
the polysaccharides depending on the environmental pH. At pH 3.0
(Fig. 6A), lentil legumin-like protein formed a-helix structure at the
interface (1652 cm1) in the presence of polysaccharides. The a-
helix conformation is energetically favorable when strong electro-
static interactions between polymers are present, contributing to
the stability of the complex (Giroda, Longchambon, Begub, Tourne-
Petheil, & Devoisselle, 2004). b-Sheets (1635 and 1684 cm1) and b-
turns (1669 cm1) were also identified. The absorption at 1618-
1620 cm1 suggests formation of protein aggregates at the interface
for protein alone and with polysaccharides. Presence of aggregated
structures in coacervates has been observed by Wang, Li, Wang, Lal,
and Huang (2007) in b-lactoglobulin-pectin coacervates. At pH 5.0
(Fig. 6B), a-helix structures (1652e1656 cm1) were more promi-
nent in the protein-polysaccharide foams than in the protein foam.
This molecular configuration might have contributed to the better
stability of protein-polysaccharide foams than at other pH values
(Fig. 5B). a-Helices may be better packaged side by side, since they
are adsorbed with their long axis parallel to the air/liquid interface,
which would allow the side chains that protrude to interdigitate
creating more non-covalent intramolecular interactions leading to
an improved surface networks (Lavigne, Tancre de, Lamarche, &
Max, 1992).
The molecular conformation of the protein at the foam interface
suffered a remarkable change at pH 7.0 (Fig. 6C) that could partially
explain the low stability of lentil legumin-like protein-poly-
saccharide foams at this pH. The protein lost its a-helix structure
(1651 cm1) in the presence of polysaccharides and the protein in
foams containing pectin and guar gum gained unordered structures
(1640 cm1). Such changes might partially explain the lower
resistance of the protein-polysaccharide foams against collapse
because of the increased intra-protein flexibility produced by un-
ordered structures that led to a less compact structure and relaxed
interface (Cascao-Pereira, Theodoly, Blanch, & Radke, 2003).
3.4.2. Foam microstructure by CLSM
CLSM was applied to visualize the distribution of protein (red)
and polysaccharide (cyan) in the foams by staining these two
components with fluorescence dyes as shown in Fig. 7. The images
confirm both the occurrence of segregative phase separation at pH
7.0 and the formation of coacervates at pH 3.0, especially in the
presence of xanthan and pectin. Also, it was possible to observe the

Fig. 7. CLSM images of the lentil legumin-like protein-polysaccharides foams at pH 3.0. (A, B, C, D), 5.0 (E, F, G, H), and 7.0 (I, J, K, L): lentil legumin-like protein, (1st column): with
guar, (2nd column): with xanthan (3 d column), and with pectin (4th column). P: Lentil legumin-like protein; G: Guar gum; P-G: Lentil legumin-like protein and guar gum; Pec:
Pectin; P-Pec: Lentil legumin-like protein and pectin; X: xanthan gum; P-X: Lentil legumin-like protein and xanthan gum.
912 M. Jarpa-Parra et al. / Food Hydrocolloids 61 (2016) 903e913
thin layer of protein film around the bubbles and how its structure
was changed by the presence of the polysaccharides at different pH
values. At pH 3.0 (Fig. 7B, C, D), the surface membrane of the
bubbles appeared smooth and homogeneous. The coacervates are
visible in the lamellae area of all the foams (marked by circles in the
micrographs). Such coacervate exhibiting gel-like interfacial
structure could explain the increased viscosity of the foam (Fig. 2A)
(Guillermic et al., 2009; Salonen et al., 2010). Interestingly, the
presence of the coacervates did not alter the smoothness of the
protein film layer around the foam bubble. Instead, they seem
anchored to the protein membrane layer and connected with each
other, building a thick network, avoiding the bubbles coalescence.
At pH 5.0 (Fig. 7F, G, H), aggregates also appeared in the lamellae of
all foams. Some of the aggregates are on the surface of the bubbles,
adding thickness to the bubble membrane, while others remained
in the Plateau borders. The latter would likely be responsible for the
high viscosity determined in the protein-guar gum and protein-
pectin foam (Fig. 2B) (Guillermic et al., 2009; Salonen et al.,
2010). The jamming effect of aggregates can be also confirmed in
these micrographs (Fig. 7F and H) as delineated by the encircled
zones, where the coacervates in the Plateau borders are confining
the liquid trapped in between. The presence of the aggregates again
did not interrupt the smoothness of the protein membrane layer in
the lamellae of pectin and guar containing foams. For the protein-
xanthan foam, it seems that the thin layer of film around bubble
surface was broken in some points compromising the interfacial
network integrity (Fig. 7G). This may suggest an incipient phase
separation occurring at this pH in the presence of xanthan gum due
to its higher charge density (Fig. 1E). At pH 7.0, the film around the
bubbles of all protein-polysaccharide foams seem fragmented. A
close look at the inserts shows that a phase separation was occur-
ring at the interfacial layer and this caused a disruption of the
protein film around the bubbles, which explained the poor foam
stability at this pH (Sengupta & Damodaran, 2000). The protein
seems partially displaced through the formation of islands of both
polymers with the protein being compressed into regions of
increasing thickness. This gradual displacement of the protein by
the polysaccharides can be explained by the orogenic mechanism
(Murray, 2007) in which there is increasing concentration of the
other component at the interface until the protein network breaks
and proteins are displaced. Such orogenic mechanism was
confirmed in protein-surfactant and protein-protein foams as re-
ported by Murray (2007).
4. Conclusions
Protein-polysaccharide interactions due to pH variation had a
significant impact on the foam properties stabilized by the lentil
legumin-like protein-polysaccharide mixtures. The foam mean life
value at pH 5.0 was dramatically increased from 51 min to 126, 117,
and 275 min when guar, xanthan, and pectin were added into the
protein system, respectively. The proximity to pI favored formation
of protein-polysaccharide aggregates that could be adsorbed onto
the bubble/water interfacial layer to form stiff interfacial networks,
hindering bubbles coalescence and thus, coarsening of the foams.
In addition, such aggregates plugged the junctions of the Plateau
borders and slowed down the drainage by a jamming effect. All
these contributed to the greatly improved foam stability. The
addition of polysaccharides also significantly improved foam sta-
bility at pH 3.0, with the protein-xanthan gum system being the
best (mean life of 87 min). At this pH, the associative interactions
led to a coacervation phenomenon. The presence of these co-
acervates may have stabilized the foams against collapse due to
the formation of an electrostatically cross-linked gel-like interfa-
cial network. Also, lentil legumin-like protein underwent a
favorable conformation change at the foam interface, in the pres-
ence of the polysaccharides at pH 3.0 and 5.0. a-Helix structures
were formed in the coacervates and aggregates that might have
contributed to the better stability of the protein-polysaccharide
foams. At pH 7.0, the mean life of the foams was reduced in the
presence of all selected polysaccharides. Phase separation induced
a disruption of the protein layer around the bubbles making them
weaker and easier to break, which had a negative impact on the
foam stability. This disturbance in the protein film might be a
result of partial displacement of the protein by the polysaccharides
through an orogenic process. In addition, proteins lost their a-helix
structure, while gaining unordered structures, leading to interfa-
cial networks with reduced resistance against collapse. As foam
stability is dependent in part on the geometry and dynamic
changes of the bubbles, in a future research, a microscopy tech-
nique will be combined with image analysis, to investigate the
geometric and dynamic characteristics of bubbles in foam;
including bubble size and shape, bubble growth and movement,
and individual bubble behavior. Thus will allow a further under-
standing of the stabilization mechanism of the lentil protein-
polysaccharide complexes.
Improvement of mean life of lentil legumin-like protein foams at
mild and acidic pH using guar, xanthan, and pectin provides op-
portunities for potential applications in food products in this range
of pH.
Acknowledgments
The authors are grateful to the Natural Sciences and Engineering
Research Council of Canada (NSERC), Saskatchewan Pulse Growers,
Alberta Crop Industry Development Fund Ltd. (ACIDF) and Alberta
Innovates Bio Solutions (AI Bio) for financial support as well as
Canada Foundation for Innovation (CFI) for equipment support.
Lingyun Chen thanks the NSERC-Canada Research Chairs Program
for its financial support. Marcela Jarpa thanks CONICYT-Chile for
the support given through the scholarship Becas Chile.
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