Interpolymeric Complexes Formed Between Whey

Proteins and Biopolymers: Delivery Systems of

Bioactive Ingredients
Monique Barreto Santos, Naiara Rocha da Costa, and Edwin Elard Garcia-Rojas

Abstract: Whey proteins are obtained from dairy industry waste. Studies involving the analysis of the bioactive
compounds in whey show health benefits, as it is an excellent source of indispensable amino acids. Milk whey contains
principally β-lactoglobulin, α-lactoglobulin, bovine serum albumin, and lactoferrin, proteins with innumerable functional
and technological properties. One application of these proteins in food is the formation of interpolymer complexes,
along with other proteins or anionic polysaccharides. The formation of complexes occurs mainly through electrostatic
interactions between a negatively charged biopolymer and a positively charged biopolymer. This formation is influenced
by factors such as pH, ionic strength, and biopolymer ratio. Because they do not use high temperatures and chemical
reagents and have additional nutritional and functional value, these complexes have been used as encapsulating agents for
bioactive ingredients. Recent studies on their training and applications are addressed in this review to boost new research
and applications in the food industry, thus increasing opportunities for utilizing whey proteins.
Keywords: bovine serum albumin, coacervate complexes, electrostatic interactions, lactoglobulin, microencapsulation

Introduction The main advantage of the use of coacervate complex from

Whey protein (WP) is the result of whey utilization and is
obtained as a byproduct of the dairy industry, specifically cheese-
making, from the coagulation of casein. Until recently, whey was
massively discarded as effluent in rivers, causing significant en-
vironmental damage due to its high biological oxygen demand
(Walstra, Wouters, & Geurts, 2006). More recently, whey has be-
come of great interest to the food industry after the discovery of
the high biological value of the soluble protein fractions, which
have begun to be used as a raw material in the elaboration of new
products with special characteristics and profit opportunity (Urista,
Álvarez, Riera, Cuenca, & Téllez, 2011). Among the most studied
proteins found at the highest concentrations are β-lactoglobulin
(β-Lg), α-lactoglobulin (α-Lg), bovine serum albumin (BSA), and
lactoferrin (LF; Sgarbieri, 2004).
One alternative for the utilization of the whey proteins is in the
formation of interpolymeric complexes or coacervates through
interactions with other natural polymers (biopolymers), such as
polysaccharides and proteins, which are then used as food ingre-
dients, encapsulating agents for bioactive compounds, texturing,
and stabilizing properties (Turgeon, Schmitt, & Sanchez, 2007).
CRF3-2017-0241 Submitted 12/7/2017, Accepted 3/9/2018. Authors Santos, da
Costa, and Garcia-Rojas are with Programa de Pós-graduação em Ciência e Tecnologia
de Alimentos (PPGCTA), Univ. Federal Rural de Rio de Janeiro (UFRRJ), Rodovia
BR 465, Km 7, Seropédica/RJ, 23890-000, Brazil. Author Garcia-Rojas is also
with Laboratório de Engenharia e Tecnologia Agroindustrial (LETA), Univ. Federal
Fluminense (UFF), Av. dos Trabalhadores, 420, Volta Redonda/RJ, 27255-125,
Brazil. Direct inquiries to author Garcia-Rojas (E-mail: edwin@vm.uff.br).
whey proteins compared to complexes from other proteins is their
high added value due to the diverse nutritional and functional
properties of whey proteins added to high productivity and low
cost because it results from the reuse of a byproduct (Almeida,
Conte-Júnior, Silva, & Alvares, 2013).
When the biopolymers have the same charge, the separation
occurs in two phases each one rich in a biopolymer, known as in-
compatibility thermodynamic (segregation). However, when the
biopolymers have opposite charges, the separation occurs in one
phase rich, mainly of the biopolymers, and the other essentially
in the solvent called the thermodynamic compatibility (coacerva-
tion). In the last case, the intensity of formation can be influenced
by intrinsic characteristics, such as molecular mass, conforma-
tion, and density of charges, and extrinsic characteristics, such
as pH, ionic strength, the ratio of biopolymers, total concentra-
tion, temperature, and pressure (de Kruif, Weinbreck, & de Vries,
2004; Tolstoguzov, 1991; Turgeon et al., 2007; Turgeon, Beaulieu,
Schmitt, & Sanchez, 2003).
Several studies have proposed elucidating the formation charac-
teristics of complexes between proteins and polysaccharides from
the standpoints of thermodynamics (de Kruif at al., 2004; Tur-
geon et al., 2007), kinetics of formation (Schmitt & Turgeon,
2011), and the functional properties of complexes (Zhang, Zhong,
& Vardhanabhuti, 2014; Norton, Espinosa, Watson, Spyropoulos,
& Norton, 2015), as well as in applications as an encapsulating
agent (Weinbreck, Kruif, & Schrooyen, 2003a; Mellema, 2004;
Zhang & Mutilangi, 2013).
Due to the growing importance given to whey proteins added
to the great interest expressed by the numerous studies found in


C 2018 Institute of Food Technologists®

doi: 10.1111/1541-4337.12350 Vol. 0, 2018 r Comprehensive Reviews in Food Science and Food Safety 1
Complexes between WPI and biopolymers . . .
the literature about delivery systems of bioactive ingredients in
pharmaceutical, food, and cosmetic applications, this review pro-
poses to provide an overview of the literature published over the
last 15 years of the use of whey proteins as an encapsulating agent
of different ingredients. It will first address the characteristics of
the main whey proteins currently used and the main mechanisms,
parameters, and interactions involved during the formation of the
complexes. Then we present the applicability of whey proteins in
the formation of complexes between biopolymers and on bioac-
tive ingredient delivery systems such as oils, vitamins, pigments,
proteins, and probiotics.
Whey
Whey, also called dairy serum or serum of cheese, is a yellow-
green colored liquid obtained from the coagulation of milk in-
tended for the manufacture of cheeses, casein, or similar products
(Codex Alimentarius, 2011). It is a byproduct of significant rele-
vance in the dairy industry due to the high volume produced and
its nutritional composition (Walstra et al., 2006).
The composition of fresh whey is 94.2% water, 0.8% whey pro-
teins, 4.3% lactose, 0.5% minerals, and 0.1% fat (Almeida et al.,
2013). As with milk, whey composition may vary with diet, race,
individual, stage of lactation, management, climate, season, envi-
ronmental condition, and animal health (Park, Juárez, Ramos, &
Haenlein, 2007). The composition and type of industrially pro-
duced whey also depends on the type of coagulation to which the
casein is subjected. Sweet whey is obtained via enzymatic coag-
ulation of casein, and acid whey is obtained through coagulation
with lactic acid bacteria or the addition of acid to milk (Etzel,
2004; Walstra et al., 2006).
Traditionally, whey was considered by cheese producers as a
byproduct of manufacturing with little or no commercial value.
For a long time, it was used in animal feed or discarded directly
in the effluent (Walzen, Dillard, & German, 2002). However,
it has become an important raw material for the production
of value-added products due to the discovery of functional and
bioactive properties of its protein components (Marshall, 2004;
Urista et al., 2011). Advances in processing technology have re-
sulted in the development of various whey protein presentation
forms (Voswinkel & Kulozik, 2014). This variation is related to
the protein content present in the product. Thus, whey protein
concentrate (WPC) varies between 25% and 80% in protein con-
tent. However, whey protein isolate (WPI) is approximately 90%
protein. Whey protein hydrolyzate (WPH) is the whey isolated
after enzymatic hydrolysis and consists of broken down proteins to
produce even smaller molecules that are more easily digested and
absorbed into the bloodstream (Carunchia, Croissant, & Drake,
2005).
Whey Proteins
In general, proteins are natural polymers (biopolymers) that,
due to the chemical properties of their basic building units,
share properties common to amphiphilic and polyampholyte
molecules. They are amphiphilic because they have both a hy-
drophobic and a hydrophilic region and polyampholytes because
they contain aliphatic and carboxylic acids that may behave as a
polyacid or polyanion. These characteristics allow proteins to in-
teract with molecules with different properties (hydrophobic, hy-
drophilic, and/or positively and negatively charged), depending on
the physicochemical conditions (Croguennec, Tavares, & Bouhal-
lab, 2017; Mezzenga & Fischer, 2013).
2 Comprehensive Reviews in Food Science and Food Safety r Vol. 0, 2018
Proteins of animal origin are important in the human diet, as
they are the main source of nitrogen and essential amino acids
(Lowery, Edel, & Mcbride, 2012). The nutritional quality of a
protein will depend on numerous factors, such as composition,
digestibility, absorption, bioavailability of essential amino acids,
and total nitrogen. Digestibility is the first factor that reflects the
efficiency of protein utilization and is considered a condition of
quality (Lemon, Berardi, & Noreen, 2002; Tang, Moore, Kujbida,
Tarnopolsky, & Phillips, 2009).
Quantitatively, the principal whey proteins account for approx-
imately 20% of total bovine milk proteins. They consist primarily
of β-Lg, α-La, BSA, immunoglobulins (Ig), LF, lactoperoxidase,
glycomacropeptides, and proteose-peptone (Almeida et al., 2013;
Etzel, 2004). They have excellent amino acid composition and
high digestibility and bioavailability of essential amino acids, espe-
cially branched-chain amino acids (BCAA, leucine, isoleucine, and
valine), in addition to having good technological properties such
as solubility (Pelegrine & Gasparetto, 2003), emulsification (Gi-
rard, Turgeon, & Paquin, 2002a), gelatinization (Ngarize, Adams,
& Howell, 2005), and water retention capacity (Vidigal et al.,
2012). In addition, they have antimicrobial and antioxidant func-
tions (Balcão et al., 2013). Due to these factors, whey protein
has been used in bakery products, salad dressings, infant formulas,
and medical nutritional formulas (Jovanovi, Bara, & Ma, 2005).
Among the most studied proteins are β-Lg, α-Lg, BSA, and LF
(Sgarbieri, 2004).
β-Lg is higher in whey (60%) and has a higher BCAA content,
with a total of 162 amino acid residues. It has a molecular mass of
18.4 kDa and an isoelectric point (pI) of 5.3 (Farrell et al., 2004;
Guimont, Marchall, Girardet, & Linden, 1997). β-LG belongs
to the lipocalin family, which is characterized by a high affinity
for hydrophobic ligands that leaves them with highly allergenic
potential (Stojadinovic et al., 2012). Eleven β-LG variants have
been identified, which include A, B, C, D, E, F, G, H, I, J, and W; A
and B occur most frequently in most bovine breeds (Caroli, Chessa,
& Erhardt, 2009). In addition, the protein appears as a dimer but
dissociates into monomers when denatured (Kontopidis, Holt,
& Sawyer, 2004). It is recognized for its high technological and
functional value; however, because it is the most abundant protein
and the most allergenic and antigenic, it may present health risks
to patients allergic to cow milk proteins, especially children. This
is because its globular structure is stable against acid hydrolysis and
proteolytic enzymes present in the stomach, and thus it remains
intact after digestion (Sgarbieri, 2004; Stojadinovic et al., 2012).
The α-Lg protein represents 20% of total whey protein, con-
taining 123 amino acid residues and having the highest tryptophan
content (6%) among all dietary protein sources. It is also rich in
lysine, leucine, threonine, and cysteine. Its molar mass is 14.2 kDa
and it has a pI of 4.8 (Kinsella & Whitehead 1989; Farrell et al.,
2004). To date, three variants (A, B, and C) have been identified
(Caroli et al., 2009). Its biological properties include anticancer ac-
tivity and antimicrobial activity against pathogenic bacteria, such
as Escherichia coli, Staphylococcus aureus, and Klebsiella pneumonia,
in addition to being rich in tryptophan, a precursor of niacin
and serotonin (Haraguchi, Abreu, & Paula, 2006; Santos, Teix-
eira, & Rodrigues, 2011). Moreover, α-Lg is a metallo-protein
with a calcium atom that enables it to bind to other proteins.
Among the whey proteins, α-Lg is the most thermally stable,
because calcium promotes the formation of intermolecular ionic
bonds, which makes the molecule resistant to thermal unfolding
(Yada, 2004). Commercially, α-Lg has been widely used in in-
fant formulas due to its structural and conformational similarity to

C 2018 Institute of Food Technologists®
Complexes between WPI and biopolymers . . .
breast milk proteins, and it is also used in sports nutrition foods due
to its branched amino-acid content (Walzem, Dillard, & German,
2002).
BSA corresponds to approximately 3% of whey proteins, has 583
amino acid residues, and is rich in cysteine (6%) which has affinity
for free fatty acids and other lipids, and favors its transport in the
bloodstream. It also performs many other functions, such as elim-
ination of oxygen-free radicals and deactivation of various toxic
lipophilic metabolites such as bilirubin; it has a molecular mass of
66.4 kDa and pI 4.8 (Emerson, 1989; Kinsella & Whitehead 1989;
Farrell et al., 2004; Neemann, Rosenberger, Jefferson, & Mcadam,
2013). Among the whey proteins, BSA is the most widely studied,
particularly because of its structural similarity to human serum al-
bumin. The BSA molecule is composed of a chain organized
into three homologous helical domains, which have two subdo-
mains that share a common helical form joined by peptide bonds.
They are characterized by a small number of tryptophan residues
and a high number of cysteines. BSA has only one free cysteine
residue, and the remaining residues form 17 disulfide bonds that
help maintain the tertiary structure (Dong, Chen, & Hu, 2007;
Sinha, Radha, Prakash, & Kaul, 2007). X-ray crystallography data
showed that the structure of albumin is predominantly composed
of α-helix (67%; Bujacz, 2012; Damodaran & Fennema, 2010).
BSA can also form dimers, especially at high concentrations or in
its crystallized form (Carter & Ho, 1994).
LF accounts for approximately 1% to 2% of total whey pro-
teins, contains approximately 680 amino acid residues, and has a
molar mass of 76.1 kDa, and a pI of 8.9 (Farrell et al., 2004; Mar-
shall, 2004). It transports iron ions (Fe2+ and Fe3+ ), in addition to
other metals such as Cu2+ , Zn2+ , and Mn2+ (González-Chavez,
Arévalo-Gallegos, & Rascón-Cruz, 2009). Due to its iron bind-
ing role, LF can be found in the form of apo-lactoferrin (apo-LF)
when it is less than 5% iron-saturated, as opposed to the holo-
lactoferrin (holo-LF) form when it has higher saturation (Steijns
& Hooijdonk, 2000). Because of its high affinity to the ion, it is the
only protein capable of binding to the metal at various pH values
and in a reversible form (Wally & Buchanan, 2007). Its molecular
structure consists of a simple polypeptide chain with two highly
homologous terminal globular (C and N) lobes. These are linked
together by parts of an α-helix, with each lobe containing a bind-
ing site (Öztaş Yeşim, & Ozguneş, 2005). Each lobe consists of two
sublobes called N1, N2, C1, and C2 (Steijns & Hooijdonk, 2000).
It is considered a multifunctional protein because it plays impor-
tant roles in biological systems, such as absorption of intestinal iron
during breastfeeding of newborns; it also has antibacterial, antifun-
gal, antiviral, anti-inflammatory, and immunoregulatory activities
and performs antioxidant actions (Balcão et al., 2013).
Formation of inter-polymer complexes
Phase separation was first observed by Tiebackx (1911) in a
study of gelatin and gum acacia (GA). In 1929, Bungenberg de
Jong and Kruyt proposed the first theoretical explanation for the
phenomenon of simple coacervation. When studying complex
coacervation formation between gelatin/GA in 1949, Bungenberg
de Jong realized that the interaction occurred due to changes in the
electrostatic charges around the biopolymers (GA with negative
charge and gelatin with positive charge). It was also determined
that these changes in charge were promoted by changes in pH and
NaCl (Bungenberg de Jong, 1949; Bungenberg de Jong & Kruyt,
1929).
Overbeek and Voom (1957) reported that coacervation oc-
curred via competition between the electrostatic forces. This

C 2018 Institute of Food Technologists®
promoted the interaction between the charged molecules and
the entropic effects that tended to disperse the interaction. This
theory was based on the following assumptions: (a) molecules
have a random chain configuration, (b) solvent–solute in-
teractions are less significant, (c) interacting forces are dis-
tributed according to the nature orientation of the biopoly-
mer, and (d) there is no specific local interaction between
molecules.
Later, Tainaka (1980) developed a model to understand the for-
mation of complex coacervation that is more comprehensive than
all previous theories and is still accepted today. It is applicable
to a great number of systems containing a high and low density
of charges. According to Tainaka, the driving forces for phase
separation are electrostatics and the force of attraction between
aggregates, which become stronger when the charge density and
the molar mass of the polymers increase. However, both the in-
crease in molar mass and density of charges have a maximum and a
minimum limit that prevent coacervate formation (Tainaka, 1979,
1980).
In general, proteins and polysaccharides, when added to aque-
ous solution, may present three thermodynamic phenomena: co-
solubility, thermodynamic incompatibility, and complex coacerva-
tion (de Kruif & Tuinier 2001; Tolstoguzov, 1991). Co-solubility
occurs when intermolecular interactions are absent; this occurs
when entropy is prevailing, and in this case, a stable and homoge-
neous solution results. The total concentration of biopolymers in
this situation is below the critical limit (3% to 4%; Boland, Singh,
& Thompson, 2014; de Kruif & Tuinier, 2001). Thermodynamic
incompatibility occurs when the ionic strength is high or when
the pH of the medium is above the protein pI (Benichou, Aserin,
& Garti, 2002; Boland et al., 2014). Thus, solvent–protein interac-
tions are stronger than protein-polysaccharides, because proteins
and polysaccharides carry negative charges (Doublier, Garnier,
Renard, & Sanchez, 2000). In the case of interactions between
proteins, thermodynamic incompatibility occurs when the pH of
the medium is not between the pI of the proteins. At this point,
the two proteins have the same charge and, therefore, interactions
with the solvent are stronger (Santos, Carvalho, & Garcia-Rojas,
2018).
Coacervation is defined by the International Union of Pure
and Applied Chemistry (IUPAC; 1997) as a colloidal separa-
tion of systems in two liquid phases and occurs when biopoly-
mers exert strong electrostatic attraction to one another. These
molecules form complexes of three-dimensional structures during
charge neutralization. Highly hydrated and concentrated droplets
in biopolymers tend to coalesce to minimize energy. The phase
separation at the macroscopic level occurs in an electrically neutral
phase concentrated in the two biopolymers (coacervate) and an-
other phase essentially composed of solvent. This phenomenon is
called complex coacervation (Bungenberg de Jong, 1949; Burgess,
1990; de Kruif et al., 2004; Phillips & Williams, 2009). Complex
coacervation differs because it generally occurs at low concentra-
tions of biopolymers (<3 to 4 wt% total solids) and low ionic
strength (<400 mM), and when both molecules carry net oppo-
site electric charges (Tolstoguzov, 2007). Table 1 shows the most
recent work reported in the literature on the formation of inter-
polymeric complexes formed from whey proteins and biopoly-
mers, the main analyzes performed by the authors (zeta potential,
turbidimetry, particle size, microscopy, thermal analysis, and rheol-
ogy) and the most relevant parameters observed (Influence of pH,
molecular mass, salt, and ratio of biopolymers) on the formation of
complexes.
Vol. 0, 2018 r Comprehensive Reviews in Food Science and Food Safety 3
Complexes between WPI and biopolymers . . .

Table 1–Systems composed of whey proteins and biopolymers, the main analyses performed and the most relevant parameters.

WP Biopolymer Parameters Tests Reference

β-Lg Sodium alginate pH ITC, DLS, τ , ζ , Harnsilawat, Pongsawatmanit,
and McClements (2006)
β-Lg Pectin pH, CNaCl τ , AFM Wang, Wang, Ruengruglikit, and
Huang (2007)
BSA Chitosan pH, CNaCl DLS, MO, SANS, USANS, τ , Kayitmazer, Strand, Tribet,
G’, and G’’ Jaeger, and Dubin (2007)
β-Lg Lysozyme T, r MO, ITC, HPLC, τ , DLS Nigem et al. (2007)
BSA Hyaluronic acid pH, CNaCl , r DLS, τ Lenormand, Deschrevel,
Tranchepain, and Vincent
(2008)
β-Lg/α−Lg Chitosan pH, T τ , SDS–PAGE, DSC, SEM Lee and Hong (2009)
WPI Chitosan pH, r η, G’, and G’’, ITC, τ , Bastos et al. (2010)
β-Lg GA T, r DLS, μ, τ , MO, ITC Aberkane et al. (2010)
LF Pectin pH, T τ , DLS, DSC Bengoechea et al. (2011)
β-Lg k-carrageenan pH, r SEM, AFM, μ, SANS Jones et al. (2011)
β-Lg GA r, T ITC, DLS, μ, τ , MO, FTIR Aberkane et al. (2012)
BSA Beet pectin pH, r ζ , DLS, τ , SANS, GPC Li et al. (2012)
BSA Pectin r, CNaCl, pH τ , ζ , G’, and G’’ Ru, Wang, Lee, Ding, and Huang
(2012)
β-Lg LF pH, CNaCl , Ct, r ζ , DLS, SEC, Yan et al. (2013)
BSA Sodium alginate pH, r, Ct τ , ζ , TMP, MO Neemman et al. (2013)
BSA k-carrageenan CNaCl , pH, r τ , G’, and G’’∗∗∗ Chai et al. (2014)
LF GA CNaCl , CT , pH, r, T τ , ζ , XRD, AFM Gulão et al. (2014)
β-Lg LF pH, r, CNaCl τ, ζ Anema and de Kruif (2014)
BSA/ β-Lg Hyaluronic acid pH, CNaCl τ , ITC, SEC, CLSM Du, Dubin, Hoagland, and Sun
(2014)
β-Lg Pectin pH, r, kind of salt, CNaCl τ , ζ , scattering intensity Hirt and Jones (2014)
WPI Arabinogalactan-protein CNaCl , CT , pH, r, τ , ζ , DLS, MO, G’, and G’’ Wee et al. (2014)
Lactalbumin Xanthan gum and pectin pH, CT , CNaCl τ , MEV Zuvanov, Garcia-Rojas, Souza, de
Gulão, and Pereira (2014)
BSA k-carrageenan pH, r, CNaCl τ , MEV, G’, and G’’, XRD Souza et al. (2015)
WPI GA pH, r SDS-PAGE, τ , ELISA Ach et al. (2015)
BSA Linseed flour pH, r, CNaCl τ , ζ , DLS, μ Liu et al. (2015)
WPI Pectin pH, CNaCl MO, DLS, τ , δ Thongkaew et al. (2015)
β-Lg Pectin r ITC, CD, IFS Xu, Melton, Jameson, Williams,
and McGillivray (2015)
LF Sodium alginate pH, r FTIR, CD, μ, ζ , DSC Bokkhim, Bansal, Grøndahl, and
Bhandarim (2015)
β-Lg LF pH, r τ , DLS, ITC Flanagan et al., 2015
β-Lg Lysozyme pH, r ζ , DLS, τ , TEM, SEM, HPLC Diarrassouba et al. (2015)
β-Lg LF pH, r, Ct, CNaCl τ , ITC, MO Tavares et al. (2015)
α-Lg Carboxymethyl-dextran pH μ, ζ , τ Du, Reuhs,and Jones Du, Reuhs,
and Jones, 2016)
β-Lg Gum of Persia pH, r, Ct, tipo de sal, T, ITC Hadian et al. (2016)
força iônica, T
BSA/ β-Lg Gelatine pH, r, T SANS, G’ and G’’ Pathak, Rawat, Aswal, and
Bohidar (2016)
LF Casein pH, r, ionic strength τ , ζ , DLS Anema and de Kruif, (2016)
LF Pea protein pH, r τ , ζ , DLS, SAXS, AFM Adal et al. (2017)
WPI Tamarind seed mucilage pH, r ζ , τ , FTIR, XRD, MO, SEM, González-Martı́nez et al. (2017)
DSC, TGA
LF Sodium alginate pH, r τ , ζ , yield, SEM, digestion Wang, Blanch, Barro, and
in vitro, SDS-PAGE, CD, Adhikari (2017)
DPPH,
WPI Flaxseed pH, r τ , ζ , G’, and G’’, MO Liu, Shim, Shen, Wang, and
Reaney (2017)
WPI Pectin pH, T τ , ζ , DLS Zeeb, Mi-Yeon, Gibis, and Weiss
(2018)
BSA Ovalbumin pH, r, CNaCl τ , ζ , DLS, SEM, MO, DSC, Santos et al. (2018)
FTIR

Interactions between whey proteins and biopolymers
In thermodynamically compatible systems, whey proteins and
polysaccharides have opposite charges, leading to electrostatic.
However, in some systems, depending on the biopolymer used,
other interactions such as hydrogen bond van der Waals, or hy-
drophobic interactions may occur (Boland et al., 2014; Doublier
et al., 2000). Complexation is favored thermodynamically by the
enthalpy gain from the formed electrostatic interactions. These
interactions are stronger when the pH of the system is below
the pI of the protein, especially when there is low ionic strength
(Schmitt, Sanchez, Desobry-Banon, & Hardy, 1998). When the
protein–polysaccharide complexes are induced electrostatically it
is assumed that a stabilization should be achieved through sec-
ondary hydrogen bonds or hydrophobic interactions (Doublier
et al., 2000).
The hydrophobic interactions are important for the biopolymers
that have nonpolar groups in aqueous solutions and are manifested
by the association of the nonpolar groups (McClements, 2006).
Proteins have hydrophobic groups, which when exposed due to
structural modifications can interact in a nonpolar manner. The
increase in temperature or addiction of the small amounts of salt,

4 Comprehensive Reviews in Food Science and Food Safety r Vol. 0, 2018 

C 2018 Institute of Food Technologists®
Complexes between WPI and biopolymers . . .
for example, contributes to this type of interaction (Boland et al.,
2014).
By characterizing the interaction between β-Lg and GA,
Aberkane et al. (2012) verified two steps of interaction using
isothermal titration calorimetry (ITC) at pH 4.5 and 25 °C. The
authors suggest that the first step would be related to electrostatic
and hydrogen interactions for having been enthalpically driven,
and that the second step would have been driven by hydropho-
bic contributions related to conformational changes due to the
favorable binding entropy. In the studies of Gulão, Souza, da Silva,
Coimbra, and Garcia-Rojas (2014) aggregates were formed be-
tween LF and GA at 80 °C, possibly indicating the participation
of hydrophobic interactions by exposing the hydrophobic groups
of the protein after a conformational change promoted by the
increase in temperature.
Hydrogen bonds may occur between polysaccharide ester
groups and the hydroxyl, amide and carboxylic groups of the
proteins, being negatively influenced by the presence of urea and
high temperatures occurring generally at pHs above the pI of the
protein where the density of charges is lower (Girard, Turgeon, &
Gouthier, 2002b).
The van der Waals forces, however, are extremely weak elec-
trical attractions, because they promote temporary interactions of
dipoles. However, they may influence macromolecule interactions
in addition to electrostatic forces (Damodaran & Fennema 2010).
Liu, Shim, Wang, and Reaney (2015) studied the influence of
urea on the formation of complexes between BSA and flax gum,
using turbidimetric analysis data to verify the participation of hy-
drogen bonds in the process. The authors observed a reduction
in turbidity values and concluded that the electrostatic interaction
between the biopolymers primarily directed the initial stage of
formation of the complexes. Afterwards, hydrogen bonds served
to stabilize them. Tavares, Croguennec, Hamon, Carvalho, and
Bouhallab (2015) characterized the interaction between β-Lg and
LF using ITC, and they verified that the interactions were exother-
mic. This indicates the predominance of electrostatic interactions,
as they are generally encountered during protein complexation
and in oppositely charged polyelectrolyte systems. In a study that
evaluated the effect of temperature on complexes between β-Lg
and Persian gum, Hadian et al. (2016) demonstrated that elec-
trostatic interactions, hydrogen bonding, and hydrophobic inter-
actions were involved in the complexation process. The authors
also concluded that the enthalpy of binding obtained by ITC was
independent of the temperature in the studied range (25, 40, and
55 °C). In addition, they found that the temperature increase from
25 to 40 °C increases the significance of hydrophobic interactions
in complex formation. The hydrogen bonds are also weakened,
giving rise to hydrophobic interactions and exposing nonpolar
groups.
Fourier transform infrared spectroscopy (FTIR) can be used to
verify the participation of hydrogen bonds. González-Martı́nez
et al. (2017) characterized coacervate complexes formed between
WPI and tamarind gum. They observed a shift in the band rep-
resenting the stretching of N–H and O–H groups, indicating the
presence of hydrogen bonds, and therefore concluded that the hy-
drogen bonds represent a significant force in the stability of the
protein–polysaccharide complexes. Likewise, Santos, de Carvalho,
and Garcia-Rojas (2018) verified the participation of electrostatic
interactions in the reduction of bands related to amides and the
participation of hydrogen bonds in the reduction of bands repre-
senting the stretching of N–H and O–H groups in their study of
heteroprotein complexes between BSA and lysozyme.

C 2018 Institute of Food Technologists®
Parameters that influence the formation of complexes
between whey proteins and biopolymers
Numerous studies have highlighted both the conditions of the
medium, such as pH and ionic strength, and the physicochemical
characteristics of the polymers, charge density, and molar mass, as
parameters capable of controlling the coacervation process (Wein-
breck, de Vries, Schrooyen, & de Kruif, 2003b).
pH
The interactions between whey proteins and polysaccharides
are directly controlled by the degree of ionization of the biopoly-
mers, and thus the pH of the medium is important in electrostatic
control (Turgeon et al., 2007). For each pair of cationic/anionic
biopolymers, there is a pH at which the charge density will be
maximal and equivalent and, consequently, the intensity of the
electrostatic interactions and formation of coacervates will also be
maximal (Phillips & Williams 2009). These interactions generally
occur at a pH below the pI of the protein and above the pKa of
the polysaccharide (Doublier et al., 2000).
The complex formation mechanism can be evidenced by tur-
bidimetric techniques, zeta potential, and particle size (Weinbreck
et al., 2003b). From the turbidimetric titration of the basic pH
to the acidic pH of a system composed of protein and polysac-
charide, the following stages of formation can be evidenced:
(a) pH > pI, protein and polysaccharide have a high negative
charge density that promotes the repulsion between them; (b) the
pH is reduced to a critical value (pHc ), and there is a slight increase
in turbidity due to the weak association between the polymers,
forming soluble complexes; (c) with further acidification of the
system, the turbidity increases abruptly due to the association of
the protein and polysaccharide forming the insoluble or coacer-
vate complexes, denoted as pHθ 1 ; (d) when the pH is reduced to
values close to the polysaccharide pKa , its anionic groups lose neg-
ative charge density, weakening the interactions and dissociation
of the complex, denoted as pHθ 2 .
The turbidity variation through acid titration in a system con-
taining BSA and k-carrageenan was studied. As the pH decreases,
there is a decrease in electrostatic repulsion and a slight increase in
the turbidity of the system due to the formation of soluble com-
plexes (pHc ). This is followed by a remarkable increase in turbidity
due to the increase in the size and number of complexes formed
between BSA and k-carrageenan, which occurs up to pHmax , with
the maximum formation of coacervate complexes (Chai, Lee, &
Huang, 2014). In the pH range above the pI of the protein, the
biopolymers begin to carry similar charges of sufficient magnitude
to favor the separation of phases, and thermodynamic incompat-
ibility occurs. This behavior was reported by Thongkaew, Hin-
richs, Gibis, and Weiss (2015) when studying the charge density
of whey protein and pectin as a function of pH. At pH 3.2, the
pectin presents negative charges, whereas the protein is positively
charged, favoring an interaction between them. However, the val-
ues obtained at pH 6.5 for the zeta potential for both are negative,
confirming strong repulsion between the two polymers.
The pKa of polysaccharides can determine the limit of com-
plex formation. According to Bengoechea, Jones, Guerrero, and
McClements (2011), sediments formed during titration of LF
and pectin are solubilized at pH 2.0. From this pH, the pectin
molecules lose most of their negative charge, and the electrostatic
interactions are weaker. In the acid titration between LF and GA,
Gulão, de Souza, da Silva, Coimbra, and Garcia-Roja (2014) re-
ported a reduction of turbidity from pH 3.0 and total solubilization
of the sediments at pH 2.0, called pHθ 2 .
Vol. 0, 2018 r Comprehensive Reviews in Food Science and Food Safety 5
Complexes between WPI and biopolymers . . .
Figure 1–Graphical representation of the turbidity variation as a function
of pH of a system composed of BSA and lysozyme. The turbidity value has
a small increase from pHc followed by a sharp rise in pHθ1 , to maximum
value (pHmax ) with subsequent reduction in pHθ2 (Adapted Santos et al.,
2018).
Unlike the authors cited above, Liu et al. (2015) reported a pHc
above the protein pI for the BSA solution and flaxseed gum at
the ratio of 1:1. The formation of soluble complexes occurred
due to the interaction of a few cationic groups of BSA with the
anionic groups of the gum. Following acidification, the pHθ 1
found was 4.8, just below the pI of the BSA (4.9). In the same
study, the authors discussed the results obtained using dynamic
light scattering (DLS), which determines particle size, and they
verified the same sizes found for solutions of biopolymers in the
mixture at pH above pHc , suggesting no formation of complexes.
However, at pH 5.0 > pHθ 1 , the observed particle sizes suggested
the formation of soluble complexes, and at pH 4.0 < pHθ 1, particle
sizes matched the formation of insoluble complexes.
Similar to the formation of complexes between proteins and
polysaccharides, the formation of heteroprotein complexes is dis-
tinguished by the formation of complexes at pH values between
the pI of the proteins. In this case, one protein behaves as a neg-
atively charged anionic biopolymer (below its pI), and the other
behaves as a positively charged cationic biopolymer (above its pI;
Santos et al., 2018). The effect of pH on heteroprotein complex
formation between β-Lg and lysozyme was studied by Diarras-
souba et al. (2015) through turbidimetric analysis and zeta poten-
tial. In this study, turbidity was observed due to the formation of
complexes over a wide pH range (6.8 to 10.0). At pH 5.0 and
11.0, near the pI of β-Lg (5.3) and lysozyme (10.7), respectively,
this increase was not verified. These results are justified because
at pH 6.8, 7.5, and 8.0, the proteins carry negative (β-Lg) and
positive (lysozyme) charges, which allow their interaction. Ana-
lyzing zeta potential data, the authors observed that the pH with
highest turbidity coincided with the lowest zeta potential value.
This is because there is greater interaction and equilibrium of the
charges, as the supercritical charge is closer to 0 mV.
A similar study was performed by Santos et al. (2018) using BSA
and lysozyme. The complexes formed in the pH range between
8.0 and 11.0, presenting maximum complexion at pH 9.0. Figure 1
shows the data obtained by the turbidimetric analysis at a given
ratio. In this Figure, the three sequential formation processes are
represented: pHc , representing the beginning of the formation of
the soluble complexes; pHƟ1 , which determines the formation
of the insoluble complexes; and pHƟ2 , which determines the end
of the process, indicating the co-solubility of the biopolymers.
6 Comprehensive Reviews in Food Science and Food Safety r Vol. 0, 2018
Ionic strength
The coacervation process is influenced by the addition of salt
and is generally verified by the addition of NaCl. Low concentra-
tions may favor the formation of complexes to a critical level where
attractive forces between polymers are diminished (Li et al., 2012;
Weinbreck et al., 2003b). Under these conditions, salt favors ac-
cess to polymer charges, as it “unwinds” the molecule, facilitating
electrostatic attraction (Weinbreck et al., 2003b). However, at high
salt concentrations the competition created by the Na+ ions by
the negative sites of the polysaccharide molecule, and the Cl− ions
by the positively charged sites of the protein, shield the biopoly-
mer charges, decreasing the interaction between them (Schmitt,
Aberkane, & Sanchez, 2009). The ionic force acts by covering
the coulombic forces between the oppositely charged biopoly-
mers which can completely suppress the complexation by having
an effect on the Debye length (Dubin, Gao, & Mattison, 1994).
As reported by previous work (Gulão et al., 2014; Gurov,
Gurova, Leontiev, and Tolstoguzov, 1988) also found that low salt
concentrations (100 to 250 mM NaCl) favored interactions be-
tween lactoferrin and gum arabic by observing increased turbidity
with increased particle size; 250 mM was the largest diameter. In
contrast, a decrease in particle diameter correlated to the turbidity
data, indicating its negative influence on the interaction at higher
salt concentrations (300 and 500 mM NaCl). Similar results were
observed by Laos, Brownsey, and Ring (2007) when studying the
complexation between furcellaran gum and BSA and/or β−Lg.
They reported that the best salt concentration for the process was
30 mM. Liu et al. (2015) found that the addition of salt (NaCl)
in the system containing BSA and flaxseed gum not only reduces
the formation of the complex to half in the concentration of 50
mM but also alters the values of pHc and pHθ 1 . In this study,
they observed a decrease in pHc values from 5.4 to 4.8 and from
pHθ 1 from 5.0 to 4.4, leading to a decrease in the number and
stoichiometry of coacervate complexes formed.
In studies of complexation between β-lactoglobulin and pectin
proposed by Sperber, Stuart, Schols, Voragen, and Norde (2010)
the results regarding the influence of ionic strength are in agree-
ment with those previously mentioned. The authors observed that
the increase in ionic strength reduced the enthalpy contribution
of the interaction, concluding that the addition of NaCl inhibits
the electrostatic interactions fundamental to complexation.
Hadian et al. (2016) studied the influence of concentration (0 to
100 mM) and the type of ions (Na+1 and Ca+2 ) on the formation
of complexes between β-Lg and the water-soluble fraction of
Persian gum (WPG). Salt concentrations of 50 mM (NaCl) and 15
mM (CaCl2 ) were sufficient to promote a significant reduction of
coacervation. In addition, the authors concluded that the effects
of divalent ions (Ca+2 ) were higher than those of monovalent
ions (Na+1 ) by suppressing complex coacervation at lower ionic
strength values. According to Schmitt et al. (2009), this behavior
is due to the double gain in entropy after the release of two
monovalent ions compared to a single divalent ion. Moreover, the
addition of mono- and divalent ions shift critical values of pH
to more acidic values to compensate for the partial blockade of
charges induced by the additional ions; that is, the charge density
of the proteins must increase to form the same amount of soluble
complexes or comparable neutralization of charges (Hadian et al.,
2016; Schmitt et al., 2009).
In addition to the pH shift’s relationship to complex formation,
the Debye length (Rd), a scale associated with the length of ionized
groups on the surface of the molecule, can be related to changes
in pHc and pHθ (Orme & Giocondi, 2007). Chai et al. (2014)

C 2018 Institute of Food Technologists®
Complexes between WPI and biopolymers . . .
associated this parameter with distance from the protein to the
polysaccharide, and the electrostatic attraction between the protein
and the polysaccharide is suppressed when the distance between
them is greater than the Debye length. The addition of salt to the
BSA and k-carrageenan system suppressed electrostatic interactions
between them, thus displacing pHc , pHθ , and pHmax .
The salt effect was also analyzed for heteroprotein complexes.
The influence of NaCl on complex formation between β-Lg and
LF was reported by some authors. Yan et al. (2013) and Tavares
et al. (2015) found similar results, where the maximum ionic
strength for coacervation was ࣘ20 mM; unlike Anema and de
Kruif (2014), who found coacervate formation at ࣘ100 mM. Ac-
cording to Tavares et al. (2015), results may vary depending on
the source variation and quality of the protein. These can include
the degree of iron saturation of the LF, which may affect flexi-
bility and the ability to coacervate, the presence of denatured or
aggregative complexes, or even the type of coacervation medium.
Coacervation generally occurs in aqueous media with relevant pH
variation during protein mixing, which alters the ionic equilib-
rium kinetics. Santos et al. (2018) also evaluated the influence of
NaCl on the formation of heteroprotein complexes between BSA
and lysozyme, and they observed that, for minimal concentrations
of salt (<10 mM), the interaction was reduced.
Molecular mass and ratio of biopolymers
In the coacervation phase, the molecular agglomerates overlap
each other and increase in ionic density, resulting in an electro-
static energy gain. The forces governing this separation are propor-
tional to molecular mass and charge density (Burgess, 1990). The
molecular mass corresponds to the volume that the biomolecule
occupies, and thus a larger volume leads to more interactions that
can lead to formation of complexes (Phillips & Williams, 2009).
According to Weinbreck, Tromp, and de Kruif (2004a), additional
positively charged proteins are required to compensate for the
negative charges of the polysaccharide at higher pH values. The
protein becomes more charged and the polysaccharide less charged
at lower pH, and thus fewer protein molecules will be required for
charge-balancing.
In studies by Ach et al. (2015), the pH of higher yield of com-
plex formation increased positively as the ratio between α-Lg
/β-Lg proteins and GA increased. This yield was measured from
the protein concentration of the supernatant. In this same study,
the authors compared α-Lg/β-Lg behavior in the coacervation
process. First, maximum coacervation occurred at various pH val-
ues according to their pI, that is, pH 4.5 for β-Lg and pH 3.4
for α-Lg. Next, the authors concluded that the interaction was
more intense with β-Lg than with α-Lg, according to the residual
concentration of protein in the aqueous phase at maximum pH.
Based on these results and comparing the characteristics of each
protein, they suggested that the molar mass difference and chemi-
cal structure were mutually responsible for the interaction affinity
difference (Ach et al., 2015).
In the case of heteroprotein complexes, the difference in size
between protein pairs (acidic and basic) is a key element that
defines both the optimal pH and the formation ratio (Desfougeres,
Croguennec, Lechevalier, Bouhallab, & Nau, 2010). Anema and
de Kruif (2014) found complexes with a constant stoichiometry
for LF (18.3 kDa): β-Lg (80 kDa) of 1:3 at all pH values due to the
regularization of the charge. In contrast to Yan et al. (2013), they
found a better ratio of 1:1 for the same biopolymers. Diarrassouba
et al. (2015) observed higher turbidity at a β-Lg: lysozyme ratio
of 2:1, in favor of β-Lg which is larger than lysozyme (14 kDa).

C 2018 Institute of Food Technologists®
Santos et al. (2018) found higher turbidity in a 1:2 ratio of BSA:
1ysozyme, and BSA (66 kDa) is a larger protein than lysozyme.
Temperature
Temperature may alter the conformation of proteins and
polysaccharides, favoring nonelectrostatic interactions. At low
temperatures, hydrogen bonds are favored, whereas at higher tem-
peratures hydrophobic interactions take precedent due to the ex-
posure of the binding sites after denaturation of the globular pro-
teins, as well as conformational rearrangement of the structure of
polysaccharides (Schmitt et al., 1998; Mc Clements, Decker, Park,
& Weiss, 2009; Phillips & Williams, 2009).
Nigem, Croguennec, Renard, and Bouhallab (2007) evaluated
the effect of temperature on the interaction between two proteins.
The authors concluded that the interaction between α-Lg and
lysozyme occurred both at high (45 °C) and at low temperature
(5 °C), leading to the formation of precipitates. Reversible aggre-
gates were formed at 5 °C, whereas coacervates were obtained at
45 °C. According to the authors, the formation of coacervates is
influenced by temperature because they are dependent on both
electrostatic interactions and the conformational change that oc-
curs in the proteins above 27 °C. In addition, the capacity of
aggregates formed at 5 °C to be converted to coacervation at
45 °C is due to the equilibrium of several forces involved in the
process (hydrogen, electrostatic, and hydrophobic interactions).
Aberkane, Jasniewski, Gaiani, Scher, and Sanchez (2010) inves-
tigated the effect of temperature (25 to 50 °C) to verify whether
hydrophobic interactions and hydrogen bonds play a significant
role in the complexation of β-Lg and GA. This was also done
to calculate the heat capacity variation ( Cp ), which was highly
sensitive to interactions between macromolecule residues and sol-
vent molecules. The authors verified that at 25 and 35 °C an
exothermic–endothermic sequence was recorded during the titra-
tion, indicating that the first stage was under the control of en-
thalpy and the second of entropy. In contrast, at 45 and 50 °C,
the isotherm exhibited an endothermic-exothermic sequence. Al-
though other factors are also responsible for the negative value in
Cp, because it is related to the proportion between polar and
nonpolar groups, according to the authors, the negative value in
the second binding stage may indicate a greater contribution of
hydrophobic interactions in the binding process compared to the
first stage where there was predominance of ionic bonds veri-
fied by positive Cp (Kayitmazer, 2017). The basis for the author’s
argument lies in the fact that removal of the contact between
the surface area of the protein and the solvent results in a negative
Cp . This behavior is particularly related to the interaction of wa-
ter molecules with hydrophobic sites. Through this correlation of
Cp with the burial of the surface area, the heat capacity provides
a link between thermodynamic data and structural information
(Perozzo, Folkers, & Scapozza, 2004).
Hadian et al. (2016) studied the interaction between β-Lg and
WPG, finding a high affinity at 25 °C. However, affinity and sto-
ichiometry both underwent changes with increasing temperature
(40 °C). They concluded that hydrophobic interactions became
more significant than hydrogen bonds with increased temperature.
Application of whey proteins as a bioactive encapsulating
agent: complex coacervation
Microencapsulation by complex coacervation is classified by
McClements as delivery systems based on biopolymers. In this
case, the basis for the construction of this system are biopoly-
mers, such as proteins and polysaccharides (McClements, 2014).
Vol. 0, 2018 r Comprehensive Reviews in Food Science and Food Safety 7
Complexes between WPI and biopolymers . . .
Its use as an encapsulation method has the advantage of simplic-
ity and does not require the use of high temperatures or organic
solvents. However, its use is limited by pH range, colloid concen-
tration, and/or electrolyte concentration based on the polymers
used (Comunian et al., 2013).
The basic procedure for encapsulation by coacervation was de-
veloped in the work of Bungenberg de Jong (1929) using gelatin
and GA. The first step is emulsification, where the core mate-
rial is added to a solution of cationic polymer under stirring.
The second is coacervation, where the anionic biopolymer is
added, followed by pH adjustment to initiate the interactions.
The obtained microcapsules have reduced mechanical resistance
due to the ionic nature of the interaction, which implies the
use of a crosslinking agent (Wang et al., 2014; Zhang, Pan, &
Chung, 2011). Finally, after obtaining a precipitate of microcap-
sules, the material is dried by atomization or lyophilization to give a
powder.
The complex coacervation microencapsulation process is illus-
trated in Figure 2. The active ingredient (Oil) is added to the
polymer 1 solution and, subsequently, the previously solubilized
polymer 2 is added and the pH is adjusted, similar to Jain, Thakur,
Ghoshal, Katare, and Shivhare (2015), who used WPI and GA as
a wall material in the encapsulation of β-carotene and glutaralde-
hyde as crosslinking agent.
Table 2 lists the most-recent studies that used coacervate com-
plexes formed between whey proteins and biopolymers as a wall
material for microencapsulation of different active ingredients in
addition to presenting the main analyses and parameters studied
and the efficiency of encapsulation obtained.
The use of complexes formed by whey protein and polysac-
charide as anionic biopolymer in the microencapsulation of hy-
drophilic and hydrophobic bioactive compounds has been pro-
posed by numerous authors. Among the bioactive compounds of
hydrophobic character microencapsulated by these complexes are
the oils, probiotic bacteria, pigments, flavoring and vitamins.
With regard to the biopolymers used in the encapsulations of
these bioactives, the pair WPI and GA had been the most studied.
Weinbreck, Minor, and de Kruif (2004b) studied the microencap-
sulation of sunflower, lemon, and orange oils by complex coac-
ervation using WPI and GA. The stability of the microcapsules
prepared via emulsion, followed by complex coacervation, was
strongly dependent on the pH of the medium. The capsules were
formed at pH 4.0, where the strength of the electrostatic inter-
action and viscosity were maximal, and the encapsulated oil yield
was greater than 80%. Lemon oil capsules were added to cheese
to assess flavor release. The authors observed that the size of the
capsules and the crosslinking process influenced the release of oil.
Larger and uncrosslinked capsules showed greater flavor release
than smaller and crosslinked capsules.
Using WPI, Eratte, Wang, Dowling, Barrow, and Adhikari
(2014) produced omega-3 rich tuna oil microcapsules by com-
plex coacervation. The authors found that the ideal pH and WPI:
GA ratio for complex coacervation were 3.7 and 3:1, respec-
tively. In addition, the authors confirmed that the drying method
also influenced oil stability, and they concluded that spray-dried
solid microcapsules showed better stability against oxidation than
lyophilized microcapsules.
Eratte et al. (2015) evaluated the interaction between probiotic
bacteria (Lactobacillus casei) and omega-3 oils co-encapsulated in
WPI and GA, as well as the effect of the drying, temperature,
and time on the oxidative stability of omega-3 and viability of
probiotic bacteria. Comparing the results of the microcapsules of
8 Comprehensive Reviews in Food Science and Food Safety r Vol. 0, 2018
omega-3 rich oil and probiotic separately, the authors were able
to conclude that there was a synergistic effect providing greater
fermentability of bacteria and greater stability of omega-3 oil in
spray-dried microcapsules. In a subsequent work, Eratte, Dowling,
Barrow, and Adhikari (2017) performed in vitro digestion (salivary,
gastric, and intestinal) of these same co-microcapsules, checking
for probiotic bacteria survival and oil release. According to the
authors, co-microencapsulation increased the viability of bacteria
during the digestion and retention of omega-3 fatty acids.
Microencapsulation of probiotic bacteria (Lactobacillus paraca-
sei) using WPI and GA complexes was also proposed by Bosnea,
Moschakis, and Biliaderis (2017); the authors evaluated the addi-
tion of the microencapsules in yogurts. They observed that en-
capsulated cells showed increased survival during storage and gas-
tric simulation compared to yogurts containing nonencapsulated
cells. In addition, it was demonstrated that the incorporation of
coacervate complexes did not significantly affect the rheological
properties of the yogurts.
Coacervate complexes formed by WPI and GA were also used in
the encapsulation of pigments. Jain et al. (2015) microencapsulated
β-carotene by complex coacervation at an optimal pH of 4.2 and
WPI: GA ratio of 2:1, obtaining more than 70% encapsulation
efficiency. The microcapsules showed better stability than free β-
carotene, and strong antioxidant activity even after 3 months of
storage.
In addition to WPI, recent studies have demonstrated not only
the formation of coacervate complexes with whey proteins but
also their use in the encapsulation of compounds. Among them,
BSA was studied in the encapsulation of pigments from the inter-
action with GA. Shahgholian and Rajabzadeh (2015) promoted
the encapsulation of curcumin using BSA and GA. For the forma-
tion of the complexes, ideal conditions were found in a BSA: GA
ratio of 2:1 and pH of 3.7 with maximum encapsulation efficiency
(92%) and particle size of 40 to 80 μm. According to the authors,
the technique is efficient and promising for its high-efficiency
encapsulation and ease of the method.
Carboxymethylcellulose (CMC) is an anionic polymer derived
from cellulose studied by some authors in the formation of coac-
ervate complexes. However, studies of microencapsulation are still
limited. The encapsulation of a flavoring (β-pyrene) by com-
plex coacervation was proposed by Koupantsis, Pavlidou, and
Paraskevopoulou (2014) using WPI and CMC. Coacervates pre-
pared in the highest proportion of WPI: CMC and β-pyrene mass
(6.99 g) were most effective. The authors achieved a maximum en-
capsulation yield of 42% and maximum encapsulation efficiency
of 37%. The morphological characteristics were also evaluated,
concluding that the formation of microcapsules with compact or
spongy structures depended on the concentration of biopolymer
and β-pyrene. The spongy structures formed at higher WPI:CMC
ratios showed a lower encapsulation efficiency and yield than com-
pact structures. The authors suggested that the void spaces of the
spongy structures would facilitate evaporation of the volatile con-
stituent.
In addition to the hydrophobic compounds, the hydrophilic
character compounds microencapsulated by complex coacerva-
tion using whey proteins reported in the literature are pigments,
proteins, and vitamins.
With positively charged biopolymer chitosan and negatively
charged BSA, Kurukjia, Norton, and Spyropoulos (2016) pro-
posed the use of such complexes in the encapsulation of hy-
drophilic compounds, using as a model fluorescein, rhodamine
B, and riboflavin. The results showed that the structure of the

C 2018 Institute of Food Technologists®
Complexes between WPI and biopolymers . . .

Figure 2–Microencapsulation process by complex coacervation.

Table 2–The most recent studies using coacervate complexes formed between whey proteins and biopolymers for microencapsulation of different
active ingredients.

Wall material Core material Tests Parameters EE (%) Reference

WPI and GA Sunflower / lemon ζ , MO, pH 4.0, r = 2:1, 5% oil – Weibreck et al. (2004b)
/ orange oil
PI and pectin Thiamine τ , ζ , DLS, MO, HPLC pH 3.5, r = 2:1 – Bédié et al. (2008)
WPI and CMC β Pyrene ζ , SEM, GC-FID pH 2.8, r = 6.99, β-Pinene 37 Koupantsis et al. (2014)
= 6.99 g
WPI and GA Tuna oil τ , ζ , CLSM, oxidative pH 3.75, r = 3:1 90 Eratte, Wang,
(ômega-3) estability, SEM, MO Dowling, Barrow, and
Adhikari (2014)
WPI and GA β Carotene DLS, ζ , CLSM, SEM, FTIR, in pH 4.2, r = 2:1 77 Jain et al. (2015)
vitro testing, estability
(time and T °C) DPPH,
BSA and GA Curcumin SEM, XRD, DSC, texture pH 3.7, r = 2:1, Curcumin 92 Shahgholian and Rajabzadeh
analysis = 5000 mg, (2015)
β-Lg and lysozyme Vitamin D3 Estability (time, UV), in vitro pH 7.5, r = 2:1, D3 110 90 Diarrassouba et al. (2015)
testing (SIF), in vivo testing μM,
WPI and GA L. casei and tuna Estability (time e T °C) pH 3.75 – Eratte et al. (2015)
oil
WPI and CMC β Pyrene FTIR, TGA, SEM pH 2.8, r = 6.99 36 Koupantsis et al. (2016)
BSA and chitosan Hydrophilic DLS, ζ , τ , MO, SEM, in vitro pH 5.0, r = 3:1, Ct = 1% 85 Kurukji, Norton, and
actives testing Spyropoulos (2016)
WPI and GA Lactobacillus Estability (time), in vitro pH 4.0, r = 2:1, Ct = 3% – Bosnea et al. (2016)
paracasei testing (SGF), rheology
β-Lg and LF Vitamin B9 ζ , DLS, MO, Estability pH 5.5, B9 0,25 mM, Cp 97 Chapeaou et al. (2017)
0.55 mM
WPI and pectin LF DLS, ζ , SEM, AFM, FTIR, DSC, pH 3.5, r = 2:1 25 Raei et al. (2017)
HPLC
WPI and pectin/GA Anthocyanin Estability (T °C), Confocal, pH 3.2, r = 1:1, 500 μL 99 Stănciuc et al. (2017)
colorimetry anthocyanin
WPI and GA L. casei and tuna In vitro testing pH 3.75 – Eratte et al. (2017)
oil
Definitions of abbreviations are shown in the Abbreviations section.

compounds influences the encapsulation efficiency. According to
the authors, the encapsulation efficiency at pH 5.0 was similar for
rhodomine B and fluorescein (65%). However, it has not been
possible to encapsulate riboflavin under the same conditions, as it
is a more basic molecule.
Stănciuc et al. (2017) compared microcapsules formed by GA
and pectin together with WPI in the encapsulation of antho-
cyanins. Significantly, the amount of encapsulated flavonoids and
the protective effect against anthocyanin degradation was higher
in pectin-containing microcapsules compared to GA.
The use of complexes formed between pectin and WPI was
also examined in the encapsulation of vitamins and proteins.
Bédié, Turgeon, and Makhlouf (2008) studied the encapsulation
of thiamine (vitamin B1 ) by low-methoxylation pectin, and Raei,
Shahidi, Farhoodi, Jafari, and Rafe (2017) studied encapsulation
of LF by high-methoxylation pectin; both observed similar re-
sults, even though the degrees of methoxylation of the pectins
were different. The authors found that the formation of com-
plexes was highly dependent on the pH of the medium, the WPI:
pectin ratio, and the acidification method, whether before or after
polymer mixing (pre-mixing and post-mixing acidification). They
concluded that the maximum formation of complexes occurred
at pH 3.5 with a 2:1 ratio, and the encapsulation efficiency was
higher for the pre-acidification method.
Recently, coacervate complexes formed through the interaction
between two different proteins have been the subject of studies for
the encapsulation of both hydrophobic and hydrophilic bioactive
compounds. Diarrassouba et al. (2015) used β-Lg and lysozyme to
encapsulate vitamin D3 using a ratio of 2:4:1 (β-Lg: D3 :lysozyme)
at pH 7.5, according to previous studies. The data obtained in-
dicated that microspheres effectively improved long-term stability
(4 °C) and reduced the photochemical degradation of vitamin D3 .
In addition, simulating the intestinal conditions, the authors ver-
ified not only that the capsules not only retained the vitamin but
that they were also released in the intestine for later absorption.
Chapeau et al. (2017) microencapsulated vitamin B9 through
heteroprotein complexes formed between β-Lg and LF at pH 5.5.
The authors compared the performance of aggregates formed at


C 2018 Institute of Food Technologists® Vol. 0, 2018 r Comprehensive Reviews in Food Science and Food Safety 9
Complexes between WPI and biopolymers . . .

low and high concentrations of protein and heteroprotein coac- Conclusion

ervates. These were formed in intermediary concentrations as Whey proteins are biopolymers with excellent nutritional, func-
models of biochargers and showed that the coacervates showed tional, and technological properties and are an attractive alternative
higher performance than the aggregates. In this study, the authors for the encapsulation of active ingredients and construction of de-
found that the optimal encapsulation occurred at 10 mg of vitamin livery systems. Due to their characteristics, they can form particles
per g of protein, with the formation of spherical or slightly ovoid with other biopolymers through complex coacervation and are
structures with an average diameter of 8 μm. used because they do not require chemical reagents or elevated
The use of crosslinking agents such as glutaraldehyde, glycerol, temperatures. Complexes between whey proteins and biopoly-
tannic acid, or transglutaminase has been investigated by some mers are formed mainly by electrostatic interactions and are there-
authors to increase the strength of capsules and, consequently, the fore governed by intrinsic and extrinsic factors, such as pH, ionic
retention time of the active ingredient. Jain et al. (2015) and Eratte strength, charge density, biopolymer ratio, and molecular mass, in
et al. (2017) studied the effect of crosslinking on capsules formed addition to the type of crosslinker and the characteristics of the
by WPI and GA. In the case of Jain et al. (2015), glutaraldehyde active ingredients and biopolymers that influence the final prop-
promoted the crosslinking of amide bonds between the amino erties of the produced capsules. The behavior of these complexes,
acid residues of WPI. The authors observed that microcapsule as well as their structure and kinetics of formation, have been
images using scanning electron microscopy (SEM) depended on studied through different analytical techniques, presenting appli-
crosslinking density and that the size and shape of the particles cability in food products. Despite these numerous works, specific
were maintained, which was successful in crosslinking because of conditions are required for certain active ingredients to obtain an
the agent. encapsulation efficiency and appropriate delivery system. Their
Transglutaminase is the most commonly used crosslinker in use as an encapsulating agent has become promising for a vari-
foods due to the greater potential for cross-linking in protein- ety of active ingredients and applications in different products and
based coacervates and its safety and commercial availability without formulations.
causing sensory alteration in the final product (Bakry et al., 2016).
According to Eratte et al. (2017), transglutaminase crosslinking Conflicts of Interest
favored retention of the active ingredient during salivary and gas- The authors declare that there are no conflicts of interest.
tric simulation. These authors reported that the smaller amount
of oil released in simulated salivary fluid (SSF) and SGF (simu- Acknowledgments
lated gastric fluid) compared to SIF (simulated intestinal fluid) can The authors thank the Nat. Council for Scientific and Techno-
be attributed to lower degradation of the WPI-GA complex by logical Development (CNPq) and the Foundation for Research
salivary and gastric enzymes. The stability of these complexes at Support of the State of Rio de Janeiro (FAPERJ) for financial
low pH (3.0) was reported to be due to the crosslinking process support.
occurring at this pH. The literature indicates that the chemical
crosslinking of protein in coacervated complexes reduces the pro- Abbreviations
teolytic activity of gastric enzymes, resulting in a slower release of AFM Atomic force microscopy
oil under simulated gastric conditions (Kosaraju, Weerakkody, & α-Lg α-lactoglobulin
Augustin, 2009). Apo-LF Apo-lactoferrin
Different crosslinkers, glycerol and tannic acid, were tested by BCAA Higher branched-chain amino acid
Koupantsis, Pavlidou, and Paraskevopoulou (2016) during the pro- β-Lg β-lactoglobulin
duction of β-pyrene microcapsules formed by WPI and CMC. BSA Bovine serum albumin
The authors verified that the morphological characteristics of the CaCl2 Calcium chloride
complexes varied as a function of the crosslinking agent used. The CD Circular dichroism
addition of glycerol also increased the encapsulation efficiency and CLSM Confocal laser scanning microscopy
amount of encapsulated β-pyrene. The authors concluded that CMC Carboxymethylcellulose
both the hydrogen and hydrophobic binding, as well as the differ- CNaCl Concentration of salt or ionic strength
ence in the molecular size of the crosslinkers, were responsible for CT Total concentration of biopolymers
the observed behaviors. DLS Dynamic light scattering
In addition to reports in the literature, a variety of patents address DPPH Antioxidant capacity
the encapsulation of ingredients from complex coacervation. The DSC Calorimetric differential scanning
microencapsulation of hydrophobic oil particles was patented by EE Encapsulation efficiency
Green and Lowell (1957) using gelatin, casein, or GA, which are FTIR Fourier transform infrared spectroscopy
the primary materials approved for application in food products. G ‘and G’ Dynamic rheometry
Rosenberg (1997) discovered the functionality of whey proteins GA Gum acacia
to microencapsulate volatile compounds from the formation of GC-FID Flame ionization gas chromatography
emulsions. A patent was published on the formation of these simple GPC Gel permeation chromatography
microcapsules, which were easy to handle, had high stability, and Holo-LF Holo-lactoferrin
protected against deterioration of the encapsulated compound. HMP High-methoxylation pectin
Microencapsulation from the complex coacervation of whey HPLC High-performance liquid chromatography
proteins was patented by Weinbreck, Kruif, and Schrooyen (2003a) HSA Human serum albumin
as an alternative to gelatin. The core materials may be vitamins, IFS Intrinsic fluorescence spectroscopy
oils, fats, medicines, or pesticides. Weak polyelectrolytes include Ig Immunoglobulins
anionic polysaccharides, such as GA, pectin, gellan gum, alginate, IUPAC International Union of Pure and Applied
CMC, among others, or cationic polysaccharides like chitosan. Chemistry

10 Comprehensive Reviews in Food Science and Food Safety r Vol. 0, 2018 

C 2018 Institute of Food Technologists®
Complexes between WPI and biopolymers . . .

ITC Isothermal titration calorimetry Anema, S. G., & de Kruif, C. G. (2016). Phase separation and composition of
LF Lactoferrin coacervates of lactoferrin and caseins. Food Hydrocolloids, 52, 670–77.
https://doi.org/10.1016/j.foodhyd.2015.08.011.
LMP Low-methoxylation pectin
Bakry, A. M., Abbas, S., Ali, B., Majeed, H., Abouelwafa, M. Y., Mousa, A.,
MO Optical microscopy & Liang, L. (2016). Microencapsulation of oils: A comprehensive review of
n Binding stoichiometry benefits, techniques, and applications. Comprehensive Reviews in Food Science
NaCl Sodium chloride and Food Safety, 15, 143–182. http://10.1111/1541-4337.12179.
pHc Region of slight increase in turbidity Balcão, V. M., Costa, C. I., Matos, C. M., Moutinho, C. G., Amorim, M.,
Pintado, M. E., . . . Teixeira, J. A. (2013). Nanoencapsulation of bovine
pHθ 1 Region of abrupt turbidity increase lactoferrin for food and biopharmaceutical applications. Food Hydrocolloids,
pHθ 2 Region of dissociation of the complexes 32, 425–431. https://doi.org/10.1016/j.foodhyd.2013.02.004.
pHmax Maximum turbidity point Bastos, D. S., Barreto, B. N., Souza, H. K. S., Bastos, M., Rocha-Leão, M.
pI Isoelectric point H. M., Andrade, C. T., & Gonçalves, M. P. (2010). Characterization of a
pKa (−log Ka ) chitosan sample extracted from Brazilian shrimps and its application to
obtain insoluble complexes with a commercial whey protein isolate. Food
r Reason Hydrocolloids, 24, 709–718. https://doi.org/10.1016/j.foodhyd.2010.03.008.
SALS Small angle light scattering Bédié, G. K., Turgeon, S. L., & Makhlouf, J. (2008). Formation of native
SANS Small-angle neutron scattering whey protein isolate–low methoxyl pectin complexes as a matrix for
SAXS Small angle X-ray scattering; hydro-soluble food ingredient entrapment in acidic foods. Food
SDS- PAGE Sodium dodecyl sulfate polyacrylamide gel elec- Hydrocolloids, 22, 836–844. https://doi.org/10.1016/j.foodhyd.2007.03.010.
Bengoechea, C., Jones, O. G., Guerrero, A., & McClements, D. J. (2011).
trophoresis Formation and characterization of lactoferrin/pectin electrostatic
SEC Size-exclusion chromatography complexes: Impact of composition, pH and thermal treatment. Food
SEM Scanning electron microscopy Hydrocolloids, 25, 1227–1232. https://doi.org/10.1016/j.foodhyd.2010.
SGF Simulated gastric fluid 11.010.
SIF Simulated intestinal fluid Benichou, A., Aserin, A., & Garti, N. (2002). Protein-polysaccharide
interactions for stabilization of food emulsions. Journal of Dispersion Science
SSF Simulated salivary fluid and Technology, 23, 93–123. https://doi.org/10.1080/01932690208984192.
T Temperature Bokkhim, H., Bansal, N., Grøndahl, L., & Bhandarim, B. (2015).
TEM Transmission electron microscopy Interactions between different forms of bovine lactoferrin and sodium
TGA Thermogravimetric analysis alginate affect the properties of their mixtures. Food Hydrocolloids, 48, 38–46.
https://doi.org/10.1016/j.foodhyd.2014.12.036.
TMP Transmembrane pressure
Boland, M., Singh, H., & Thompson, A. (2014). Milk proteins: From expression
USANS Ultra-small-angle scattering to food (2nd ed). San Diego: Academic Press.
WPG Water-soluble fraction of Persian gum Bosnea, L. A., Moschakis, T., & Biliaderis, C. G. (2017). Microencapsulated
WP Whey protein cells of Lactobacillus paracasei subsp. paracasei in biopolymer complex
WPC Whey protein concentrate coacervates and their function in a yogurt matrix. Food & Function, 8,
554–562. https://doi.org/10.1039/C6FO01019A.
WPI Whey protein isolate
Bujacz, A. (2012). Structures of bovine, equine and leporine serum albumin.
WPH Whey protein hydrolyzed Acta Acta Crystallographica. Section D, Biological Crystallography, 68,
XRD X-ray diffraction 1278–1289. https://doi.org/10.1107/S0907444912027047.
δ shear stress Bungenberg De Jong, H. B. (1949). Complex colloid systems. Colloid science,
Cp Heat capacity variation 2, 335–432. https://doi.org/10.2516/ogst/2009070.
H Enthalpy change Bungenberg De Jong, H. B., & Kruyt, H. R. (1929). Coacervation (partial
μ Electrophoretic mobility miscibility in colloid systems). Recueil des Travaux Chimiques des Pays-Bas, 32,
849–856.
η Intrinsic viscosity
Burgess, D. J. (1990). Practical analysis of complex coacervate systems. Journal
δ Shear stress of Colloid and Interface Science, 14, 227–238.
τ Turbidity https://doi.org/10.1016/0021-9797(90)90338-O.
ζ Zeta potential Caroli, A. M., Chessa, S., & Erhardt, G. J. (2009). Invited review: Milk
protein polymorphisms in cattle: Effect on animal breeding and human
nutrition. Journal of Dairy Science, 92, 5335–5352.
References
https://doi.org/10.3168/jds.2009-2461.
Aberkane, L., Jasniewski, J., Gaiani, C., Scher, J., & Sanchez, C. (2010). Carter, D. C., & Ho, J. X. (1994). Structure of serum-albumin. Advances in
Thermodynamic characterization of acacia gum−β-lactoglobulin complex Protein Chemistry, 45, 153–203.
coacervation. Langmuir, 26, 12523–12533. http://10.1021/la100705d. https://doi.org/10.1016/S0065-3233(08)60640-3.
Aberkane, L., Jasniewski, J., Gaiani, C., Hussain, R., Scher, J., & Sanchez, C.
Carunchia, W. M. E., Croissant, A. E., & Drake, M. A. (2005).
(2012). Structuration mechanism of β-lactoglobulin – acacia gum assemblies
Characterization of dried whey protein concentrate and isolate flavor.
in presence of quercetin. Food Hydrocolloids, 29, 9–20.
Journal of Dairy Science, 88, 3826–39.
https://doi.org/10.1016/j.foodhyd.2012.01.010. https://doi.org/10.3168/jds.S0022-0302(05)73068-X.
Ach, D., Briançon, S., Dugas, V., Pelletier, J., Broze, G., & Chevalier, Y.
Chai, C., Lee, J., & Huang, Q. (2014). The effect of ionic strength on the
(2015). Influence of main whey protein components on the mechanism of
rheology of pH-induced bovine serum albumin/κ-carrageenan coacervates.
complex coacervation with Acacia gum. Colloid Surface A, 481, 367–374.
LWT-Food Science and Technology, 59, 356–360.
https://doi.org/10.1016/j.colsurfa.2015.06.006.
https://doi.org/10.1016/j.lwt.2014.05.024.
Adal, E., Sadeghpour, A., Connell, S., Rappolt, M., Ibanoglu, E., & Sarkar,
A. (2017). Heteroprotein complex formation of bovine lactoferrin and pea Chapeau, A. L., Hamon, P., Rousseau, F., Croguennec, T., Poncelet, D., &
protein isolate: A multiscale structural snalysis. Biomacromolecules, 18, Bouhallab, S. (2017). Scale-up production of vitamin loaded heteroprotein
625–635. http://10.1021/acs.biomac.6b01857. coacervates and their protective property. Journal of Food Engineering, 206,
67–76. https://doi.org/10.1016/j.jfoodeng.2017.03.005.
Almeida, C. C., Conte-Júnior, C. A., Silva, A. C. O., & Alvares, T. S. (2013).
Whey protein: Composition and functional properties. Enciclopédia Biosfera, Croguennec, T., Tavares, G. M., & Bouhallab, S. (2017). Heteroprotein
9, 1840–1854. complex coacervation: A generic process. Advences in Colloid and Interface
Science, 239, 115–126. https://doi.org/10.1016/j.cis.2016.06.009.
Anema, S. G., & de Kruif, C. G. K. (2014). Complex coacervates of
lactotransferrin and β-lactoglobulin. Journal of Colloid and Interface Science, Codex Alimentarius. (2011). Codex standart for fermented milk. Codex
430, 214–220. http://10.1016/j.jcis.2014.05.036. Alimentarius Comission. Milk and milk products, (2nd ed). Codex Stand
243–2003.


C 2018 Institute of Food Technologists® Vol. 0, 2018 r Comprehensive Reviews in Food Science and Food Safety 11
Complexes between WPI and biopolymers . . .
Comunian, T. A., Thomazini, M., Alves, A. J. G., de Matos Junior, F. E., de
Carvalho Balieiro, J. C., & Favaro-Trindade, C. S. (2013).
Microencapsulation of ascorbic acid by complex coacervation: Protection
and controlled release. Food Research International, 52, 373–379.
https://doi.org/10.1016/j.foodres.2013.03.028.
Damodaran, P. K. L., & Fennema, O. R. (2010). Quı́mica de Alimentos de
Fennema (4th ed). Porto Alegre: Editora Artmed SA.
Desfougeres, Y., Croguennec, T., Lechevalier, V., Bouhallab, S., & Nau, F.
(2010). Charge and size drive spontaneous self-assembly of oppositely
charged globular proteins into microspheres. The Journal of Physical Chemistry
B, 114, 4138–4144. https://doi.org/10.1021/jp9090427.
de Kruif, C. G., & Tuinier, R. (2001). Polysaccharide protein interactions.
Food Hydrocolloids, 15, 555–563.
https://doi.org/10.1016/S0268-005X(01)00076-5.
de Kruif, C. G., Weinbreck, F., & de Vries, R. (2004). Complex
coacervation of proteins and anionic polysaccharides. Current Opinion in
Colloid & Interface Science, 9, 340–349. https://doi.org/10.1016/j.cocis.
2004.09.006.
Diarrassouba, F., Remondetto, G., Garrait, G., Alvarez, P., Beyssac, E., &
Subirade, M. (2015). Self-assembly of β-lactoglobulin and egg white
lysozyme as a potential carrier for nutraceuticals. Food Chemintry, 173,
203–209. https://doi.org/10.1016/j.foodchem.2014.10.009.
Dong, L., Chen, X., & Hu, Z. (2007). Study of the effect of cal-red on the
secondary structure of human serm albumin by spectroscopic techniques.
Journal of Molecular Structure, 846, 112–118.
https://doi.org/10.1016/j.molstruc.2007.01.034.
Doublier, J. L., Garnier, C., Renard, D., & Sanchez, C. (2000).
Protein–polysaccharide interactions. Current Opinion in Colloid
& Interface Science, 5, 202–214.
https://doi.org/10.1016/S1359-0294(00)00054-6.
Du, X., Dubin, P. L., Hoagland, D. A., & Sun, L. (2014). Protein-selective
coacervation with hyaluronic acid. Biomacromolecules, 15, 726–734.
https://doi.org/10.1021/bm500041a.
Du, J., Reuhs, B. L., & Jones, O. G. (2016). Influence of PEGylation on the
ability of carboxymethyl-dextran to form complexes with α-lactalbumin.
Food Chemintry, 196, 853–859.
https://doi.org/10.1016/j.foodchem.2015.10.021.
Dubin, P. L., Gao, J., & Mattison, K. (1994). Protein purification by selective
phase separation with solyelectrolytes. Separation & Purification Reviews, 23,
1–16. https://doi.org/10.1080/03602549408001288.
Emerson, T. E. (1989). Unique features of albumin - A brief review. Critical
Care Medicine, 17, 690–694.
Eratte, D., Wang, B., Dowling, K., Barrow, C. J., & Adhikari, B. P. (2014).
Complex coacervation with whey protein isolate and gum arabic for the
microencapsulation of omega-3 rich tuna oil. Food and Function, 5,
2743–2750. https://doi.org/10.1039/c4fo00296b.
Eratte, D., McKnight, S., Gengenbach, T. R., Dowling, K., Barrow, C. J., &
Adhikari, B. P. (2015). Co-encapsulation and characterisation of omega-3
fatty acids and probiotic bacteria in whey protein isolate–gum Arabic
complex coacervates. Journal of Functional Food, 19, 882–892.
https://doi.org/10.1016/j.jff.2015.01.037.
Eratte, D., Dowling, K., Barrow, C. J., & Adhikari, B. P. (2017). In-vitro
digestion of probiotic bacteria and omega-3 oil co-microencapsulated in
whey protein isolate-gum Arabic complex coacervates. Food Chemistry, 227,
129–136. https://doi.org/10.1016/j.foodchem.2017.01.080.
Etzel, M. R. (2004). Manufacture and use of dairy protein fractions. The
Journal of Nutrition, 134, 996–1002.
Farrell, H. M., Jimenez-Flores, R., Bleck, G. T., Brown, E. M., Butler, J. E.,
Creamer, L. K., . . . Swaisgood, H. E. (2004). Nomenclature of the
proteins of cows’ milk–sixth revision. Journal of Dairy Science, 87, 1641–74.
https://doi.org/10.3168/jds.S0022-0302(04)73319-6.
Flanagan, S. E., Malanowski, A. J., Kizilay, E., Seeman, D., Dubin, P. L.,
Donato-Capel, L., . . . Schmitt, C. (2015). Complex equilibria, speciation,
and heteroprotein coacervation of lactoferrin and β lactoglobulin. Langmuir,
31, 1776–1783. https://doi.org/10.1021/la504020e.
Girard, M., Turgeon, S. L., & Paquin, P. (2002a). Emulsifying properties of
whey protein-carboxymethylcellulose complexes. Journal of Food Science, 67,
113–119. https://doi.org/10.1111/j.1365-2621.2002.tb11369.x.
Girard, M., Turgeon, S. L., & Gauthier, S. F. (2002b). Interpolymer
complexing between β-lactoglobulin and low- and hight-methylated pectin
measured by potentiometric titration and ultrafiltration. Food Hydrocolloids,
16, 585–591.
González-Chávez, S. A., Arévalo-Gallegos, S., & Rascón-Cruz, Q. (2009).
Lactoferrin: Structure, function and applications. International Journal of
12 Comprehensive Reviews in Food Science and Food Safety r Vol. 0, 2018
Antimicrobial Agents, 33, 301, e1–301.e8.
https://doi.org/10.1016/j.ijantimicag.2008.07.020.
González-Martı́nez, D. A., Carrillo-Navas, H., Barrera-Dı́az, C. E.,
Martı́nez-Vargas, S. L., Alvarez-Ramı́rez, J., & Pérez-Alonso, C. (2017).
Characterization of a novel complex coacervate based on whey protein
isolate-tamarind seed mucilage. Food Hydrocolloids, 72, 115–126.
https://doi.org/10.1016/j.foodhyd.2017.05.037.
Green, B. K., & Lowell, S. (1957). Oil-containing microscopic capsules and method
of making them. Google Patents
Guimont, C., Marchall, E., Girardet, J. M., & Linden, G. (1997). Biologically
active factors in bovine milk and dairy byproducts: Influence on cell culture.
Critical Reviews in Food Science and Nutrition, 37, 393–410.
https://doi.org/10.1080/10408399709527780.
Gulão, E. S., de Souza, C. J. F., da Silva, F. A. S., Coimbra, J. S. R., &
Garcia-Roja, E. E. (2014). Complex coacervates obtained from lactoferrin
and gum arabic: Formation and characterization. Food Research International,
65, 367–374. https://doi.org/10.1016/j.foodres.2014.08.024.
Gurov, A. N., Gurova, N. V., Leontiev, A. L., & Tolstoguzov, V. B.
(1988). Equilibrium and non-equilibrium complexes between bovine
serum albumin and dextran sulfate—I. Complexing conditions and
composition of non-equilibrium complexes. Food Hydrocolloids, 2,
267–283. https://doi.org/10.1016/S0268-005X(88)80025-0.
Hadian, M., Hosseini, S. M. H., Farahnaky, A., Mesbahi, G. R., Yousefi,
G. H., & Saboury, A. A. (2016). Isothermal titration calorimetric and
spectroscopic studies of β-lactoglobulin-water-soluble fraction of Persian
gum interaction in aqueous solution. Food Hydrocolloids, 55, 108–118.
https://doi.org/10.1016/j.foodhyd.2015.11.006.
Haraguchi, F. K., Abreu, W. C., & Paula, H. (2006). Proteı́nas do soro do
leite: Composição, propriedades nutricionais, aplicações no esporte e
benefı́cios para a saúde humana. Revista de Nutrição, 19, 479–488.
https://doi.org/10.1590/S1415-52732006000400007.
Harnsilawat, T., Pongsawatmanit, R., & McClements, D. J. (2006).
Characterization of β-lactoglobulin–sodium alginate interactions in aqueous
solutions: A calorimetry, light scattering, electrophoretic mobility and
solubility study. Food Hydrocolloids, 20, 577–585.
https://doi.org/10.1016/j.foodhyd.2005.05.005.
Hirt, S., & Jones, O. G. (2014). Effects of chloride, thiocyanate and sulphate
salts on β-lactoglobulin–pectin associative complexes. International Journal of
Food Science and Technology, 49, 2391–2398.
https://doi.org/10.1111/ijfs.12560.
IUPAC. (1997). Compendium of chemical terminology (2rd ed). Oxford:
Blackwell Scientific Publications.
Jain, A., Thakur, D., Ghoshal, G., Katare, O. P., & Shivhare, U. S. (2015).
Microencapsulation by complex coacervation using whey protein isolates
and gum acacia: An approach to preserve the functionality and controlled
release of β-carotene. Food Bioprocess Technology, 8, 1635–1644.
https://doi.org/10.1007/s11947-015-1521-0.
Jones, O. G., Handschin, S., Adamcik, J., Harnau, L., Bolisetty, S., &
Mezzenga, R. (2011). Complexation of β-lactoglobulin fibrils and sulfated
polysaccharides. Biomacromolecules, 12, 3056–3065.
https://doi.org/10.1021/bm200686r.
Jovanovi, S., Bara, M., & Ma, O. (2005). Whey proteins-properties and
possibility of application. Mljekarstvo: Journal of Dairy Productionand Processing
Improvement, 55, 215–233.
Kayitmazer, A. B., Strand, S. P., Tribet, C., Jaeger, W., & Dubin, P. L. (2007).
Effect of polyelectrolyte structure on protein−polyelectrolyte coacervates:
Coacervates of bovine serum albumin with poly (diallyldimethylammonium
chloride) versus chitosan. Biomacromolecules, 8, 3568–3577.
https://doi.org/10.1021/bm700645t.
Kayitmazer, A. B. (2017). Thermodynamics of complex coacervation.
Advances in Colloid and Interface Science, 239, 169–177.
https://doi.org/10.1016/j.cis.2016.07.006.
Kinsella, J. E., & Whitehead, D. M. (1989). Proteins in whey: Chemical,
physical and functional properties. Advences in Foods and Nutrition Research,
3, 343–438. https://doi.org/10.1016/S1043-4526(08)60130-8.
Kosaraju, S. L., Weerakkody, R., & Augustin, M. A. (2009). In-vitro
evaluation of hydrocolloid–based encapsulated fish oil. Food Hydrocolloids,
23, 1413–1419. https://doi.org/10.1016/j.foodhyd.2008.10.009.
Kontopidis, G., Holt, C., & Sawyer, L. (2004). Invited review:
Beta-lactoglobulin: Binding properties, structure, and function. Journal of
Dairy Science, 87, 785–96.
https://doi.org/10.3168/jds.S0022-0302(04)73222-1.
Koupantsis, T., Pavlidou, E., & Paraskevopoulou, A. (2014). Flavour
encapsulation in milk proteins – CMC coacervate-type complexes. Food

C 2018 Institute of Food Technologists®
Complexes between WPI and biopolymers . . .
Hydrocolloids, 37, 134–142. https://doi.org/10.1016/j.foodhyd.2013.
10.031.
Koupantsis, T., Pavlidou, E., & Paraskevopoulou, A. (2016). Glycerol and
tannic acid as applied in the preparation of milk proteins – CMC complex
coavervates for flavour encapsulation. Food Hydrocolloids, 57, 62–71.
https://doi.org/10.1016/j.foodhyd.2016.01.007.
Kurukji, D., Norton, I., & Spyropoulos, F. (2016). Fabrication of sub-micron
protein-chitosan electrostatic complexes for encapsulation and
pH-Modulated delivery of model hydrophilic active compounds. Food
Hydrocolloids, 53, 249–260. https://doi.org/10.1016/j.foodhyd.2015.02.021.
Laos, K., Brownsey, G. J., & Ring, S. G. (2007). Interactions between
furcellaran and the globular proteins bovine serum albumin and
β-lactoglobulin. Carbohydrate Polymers, 67, 116–123.
https://doi.org/10.1016/j.carbpol.2006.04.021.
Lee, A. C., & Hong, Y. H. (2009). Coacervate formation of
α-lactalbumin–chitosan and β-lactoglobulin–chitosan complexes. Food
Reserach International, 42, 733–738.
https://doi.org/10.1016/j.foodres.2009.02.022.
Lemon, P. W., Berardi, J. M., & Noreen, E. E. (2002). The role of protein
and amino acid supplements in the athlete’s diet: Does type or timing of
ingestion matter? Current Sports Medicine Reports, 4, 214–221.
Lenormand, H., Deschrevel, B., Tranchepain, F., & Vincent, J. C. (2008).
Electrostatic interactions between hyaluronan and proteins at pH 4: How do
they modulate hyaluronidase activity. Biopolymers, 89, 1088–1103.
https://doi.org/10.1002/bip.21061.
Li, X., Fang, Y., Al-Assaf, S., Phillips, G. O., Yao, X., Zhang, Y., . . . Jiang,
F. (2012). Complexation of bovine serum albumin and sugar beet pectin:
Structural transitions and phase diagram. Langmuir, 28, 10164–10176.
https://doi.org/10.1021/la302063u.
Liu, J., Shim, Y. Y., Wang, Y., & Reaney, M. J. T. (2015). Intermolecular
interaction and complex coacervation between bovine serum albumin and
gum from whole flaxseed (Linum usitatissimum L.). Food Hydrocolloids, 49,
95–103. https://doi.org/10.1016/j.foodhyd.2015.02.035.
Liu, J., Shim, Y. Y., Shen, J., Wang, Y., & Reaney, M. J. T. (2017). Whey
protein isolate and flaxseed (Linum usitatissimum L.) gum electrostatic
coacervates: Turbidity and rheology. Food Hydrocolloids, 64, 18–27.
https://doi.org/10.1016/j.foodhyd.2016.10.006.
Lowery, L., Edel, J. F., & Mcbride, I. M. (2012). Dietary protein and strength
athletes. Strength & Conditioning Journal, 34, 26–32.
https://doi.org/10.1519/SSC.0b013e31826284d9.
Marshall, K. (2004). Therapeutic applications of whey protein. Alternative
Medicine Review, 9, 136–156.
McClements, D. J. (2006). Non-covalent interactions between proteins and
polysaccharides. Biotechnology Advances, 24, 621–625.
https://doi.org/10.1016/j.biotechadv.2006.07.003.
McClements, D. J., Decker, E. A., Park, Y., & Weiss, J. (2009). Structural
design principles for delivery of bioactive components in nutraceuticals and
functional foods. Critical Reviews in Food Science and Nutrition, 49, 577–606.
https://doi.org/10.1080/10408390902841529.
McClements, D. J. (2014). Nanoparticle- and microparticle-based delivery systems:
Encapsulation, protection and release of active compounds (1st ed). Boca Raton:
CRC Press.
Mellema, M. (2004). Complex coacervate encapsulate comprising lipophilic core.
Google Patents.
Mezzenga, R., & Fischer, P. (2013). The self-assembly, aggregation and phase
transitions of food protein systems in one, two, and three dimensions. Report
on Progress in Physics, 76, 1–43.
https://doi.org/10.1088/0034-4885/76/4/046601.
Neemann, F., Rosenberger, S., Jefferson, B., & Mcadam, E. J. (2013).
Non-covalent protein–polysaccharide interactions and their influence on
membrane fouling. Journal of Membrane Science, 446, 310–317.
https://doi.org/10.1016/j.memsci.2013.06.054.
Ngarize, S., Adams, A., & Howell, N. A. (2005). Comparative study of heat
and high pressure induced gels of whey and egg albumen proteins and their
binary mixtures. Food Hydrocolloids, 19, 984–996.
https://doi.org/10.1016/j.foodhyd.2004.12.008.
Nigen, M., Croguennec, T., Renard, D., & Bouhallab, S. (2007).
Temperature affects the supramolecular structures resulting from
α-lactalbumin−lysozyme interaction. Biochemistry, 46, 1248–1255.
https://doi.org/10.1021/bi062129c.
Norton, J. E., Espinosa, Y. G., Watson, R. L., Spyropoulos, F., & Norton,
I. T. (2015). Functional food microstructures for macronutrient release and
delivery. Food & Function, 6, 663–678.
https://doi.org/10.1039/C4FO00965G.

C 2018 Institute of Food Technologists® Vol. 0, 2018 r Comprehensive Reviews in Food Science and Food Safety 13
Orme, C. A., & Giocondi, J. L. (2007). Model systems for formation and
dissolution of calcium phosphate minerals. In P. Behrens, E. Bauerlein, & S.
Mann (Eds.), Handbook of biomineralization: Biomimetic and bio-inspired
materials chemistry (pp. 135–157). Weinheim: Wiley-VCH.
Overbeek, J. T. G., & Voorn, M. J. (1957). Phase separation in polyelectrolyte
solutions. Theory of complex coacervation. Journal of Cellular Physiology, 49,
7–26. https://doi.org/10.1002/jcp.1030490404.
Öztaş, Yeşim E. R., & Ozguneş, N. (2005). Lactoferrin: A multifunctional
protein. Advances in Molecular Medicine, 1, 149–154.
Park, Y. W., Juárez, M., Ramos, M., & Haenlein, G. F. W. (2007).
Physico-chemical characteristics of goat and sheep milk. Small Ruminant
Research, 68, 88–113. https://doi.org/10.1016/j.smallrumres.2006.09.013.
Pathak, J., Rawat, K., Aswal, V. K., & Bohidar, H. B. (2016). Hierarchical
internal structures in gelatin–bovine serum albumin/β-lactoglobulin gels
and coacervates. Journal of Physical Chemistry B, 120, 9506–12.
https://doi.org/10.1021/acs.jpcb.6b05378.
Pelegrine, D. H. G., & Gasparetto, C. A. (2003). Estudo da solubilidade das
proteı́nas presentes no soro de leite e na clara de ovo. Revista Brasileira de
Produtos Agroindustriais, 5, 57–65.
http://10.15871/1517-8595/rbpa.v5n1p57-65.
Perozzo, R., Folkers, G., & Scapozza, L. (2004). Thermodynamics of
protein–ligand interactions: History, presence, and future aspects. Journal of
Receptors and Signal Transduction, 24, 1–52.
https://doi.org/10.1081/RRS-120037896.
Phillips, G. O., & Williams, P. A. (2009). Handbook of hydrocolloids.
Cambridge, U.K.: Woodhead Publishing Ltd.
Raei, M., Shahidi, F., Farhoodi, M., Jafari, S. M., & Rafe, A. (2017).
Application of whey protein-pectin nanocomplex carriers for loading of
lactoferrin. International Journal of Biological Macromolecules, 105, 281–291.
https://doi.org/10.1016/j.ijbiomac.2017.07.037.
Rosenberg, M. (1997). Food treatment forming individual capsule. Google
Patents
Ru, Q., Wang, Y., Lee, J., Ding, Y., & Huang, Q. (2012). Turbidity and
rheological properties of bovine serum albumin/pectin coacervates: Effect
of salt concentration and initial protein/polysaccharide ratio. Carbohydrate
Polymers, 88, 838–846. https://doi.org/10.1016/j.carbpol.2012.01.019.
Santos, M. J., Teixeira, J. A., & Rodrigues, L. R. (2011). Fractionation and
recovery of whey proteins by hydrophobic interaction chromatography.
Journal of Chromatography B, 879, 475–479.
https://doi.org/10.1016/j.jchromb.2011.01.003.
Santos, M. B., de Carvalho, C. W. P., & Garcia-Rojas, E. E. (2018).
Heteroprotein complex formation of bovine serum albumin and lysozyme:
Structure and thermal stability. Food Hydrocolloids, 74, 267–274.
https://doi.org/10.1016/j.foodhyd.2017.08.016.
Schmitt, C., Sanchez, C., Desobry-Banon, S., & Hardy, J. (1998). Structure
and technofunctional properties of protein-polysaccharide complexes: A
review. Critical Reviews in Food Science and Nutrition, 38, 689–753.
https://doi.org/10.1080/10408699891274354.
Schmitt, C., Aberkane, L., & Sanchez, C. (2009). Protein-polyssacharide
complexes and coacervates In G. O. Phillips & P. A. Williams (Eds.),
Handbook of Hydrocolloids Woodhead (pp. 420–476). Cambridge, U.K.:
Publishing Ltd.
Schmitt, C., & Turgeon, S. L. (2011). Protein/polysaccharide complexes and
coacervates in food systems. Advances in Colloid and Interface Science, 167,
63–70. http://org/10.1016/j.cis.2010.10.001.
Sgarbieri, V. C. (2004). Propriedades fisiológicas-funcionais das proteı́nas do
soro de leite. Revista de Nutrição, 17, 397–409.
https://doi.org/10.1590/S1415-52732004000400001.
Shahgholian, N., & Rajabzadeh, G. (2015). Fabrication and characterization
of curcumin-loaded albumin/gum arabic coacervate. Food Hydrocolloids, 53,
249–260. https://doi.org/10.1016/j.foodhyd.2015.11.031.
Sinha, R., Radha, C., Prakash, J., & Kaul, P. (2007). Whey protein
hydrolysate: Functional properties, nutritional quality and utilization in
beverage formulation. Food Chemistry, 101, 1484–1491,
https://doi.org/10.1016/j.foodchem.2006.04.021.
Souza, C. J. F., Ramos, A. V., Câmara, P. B. S., Gulão, E. S., de Campos, M.
F., & Garcia-Rojas, E. E. (2015). Polymeric complexes obtained from the
interaction of bovine serum albumin and κ-carrageenan. Food Hydrocolloids,
45, 286–290. https://doi.org/10.1016/j.foodhyd.2014.10.023.
Sperber, B. L. H. M., Stuart, M. A. C., Schols, H. A., Voragen, A. G. J., &
Norde, W. (2010). Overall charge and local charge density of pectin
determines the enthalpic and entropic contributions to complexation with
β-lactoglobulin. Biomacromolecules, 11(12)3578–3583.
https://doi.org/10.1021/bm1010432.
Complexes between WPI and biopolymers . . .
Stănciuc, N., Turturică, M., Oancea, A. M., Barbu, V., Ioniţă, E., Aprodu, I.,
& Râpeanu, G. (2017). Microencapsulation of anthocyanins from grape
skins by whey protein isolates and different polymers. Food and Bioprocess
Technology, 10, 1715–1726. https://doi.org/10.1007/s11947-017-
1938-8.
Steijns, J. M., & Hooijdonk, A. C. (2000). Occurrence, structure,
biochemical properties and technological characteristics of lactoferrin.
British Journal of Nutrition, 84, S11–7.
https://doi.org/10.1017/S0007114500002191.
Stojadinovic, M., Burazer, L., Ercili-Cura, D., Sancho, A., Buchert, J.,
Cirkovic Velickovic, T., & Stanic-Vucinic, D. (2012). One-step method for
isolation and purification of native β-lactoglobulin from bovine whey.
Journal of the Science of Food and Agriculture, 92, 1432–1440.
https://doi.org/10.1002/jsfa.4722.
Tainaka, K. I. (1980). Effect of counterions on complex coacervation.
Biopolymers, 19, 1289–1298. https://doi.org/10.1002/bip.1980.360190705.
Tainaka, K. (1979). Study of complex coacervation in low concentration by
virial expansion method. i. salt free systems. Journal of the Physical Society of
Japan, 46, 1899–1906. https://doi.org/10.1143/JPSJ.46.1899.
Tang, J. E., Moore, D. R., Kujbida, G. W., Tarnopolsky, M. A., & Phillips, S.
M. (2009). Ingestion of whey hydrolysate, casein, or soy protein isolate:
Effects on mixed muscle protein synthesis at rest and following resistance
exercise in young men. Journal of Applied Physiology, 107, 987–992.
https://doi.org/10.1152/japplphysiol.00076.2009.
Tavares, G. M., Croguennec, T., Hamon, P., Carvalho, A. F., & Bouhallab, S.
(2015). Selective coacervation between lactoferrin and the two isoforms of
β-lactoglobulin. Food Hydrocolloids, 48, 238–247.
https://doi.org/10.1016/j.foodhyd.2015.02.027.
Tiebackx, F. W. Z. (1911). Gleichzeitige Ausflockung zweier Kolloide.
Kolloide Zeitschrift, 8, 198–201.
Thongkaew, C., Hinrichs, J., Gibis, M., & Weiss, J. (2015). Sequential
modulation of pH and ionic strength in phase separated whey protein isolate
– Pectin dispersions: Effect on structural organization. Food Hydrocolloids,
47, 21–31. https://doi.org/10.1016/j.foodhyd.2014.11.006.
Turgeon, S. L., Beaulieu, M., Schmitt, C., & Sanchez, C. (2003).
Protein–polysaccharide interactions: Phase-ordering kinetics,
thermodynamic and structural aspects. Current Opinion in Colloid & Interface
Science, 8, 401–414. https://doi.org/10.1016/S1359-0294(03)00093-1.
Turgeon, S. L., Schmitt, C., & Sanchez, C. (2007). Protein–polysaccharide
complexes and coacervates. Current Opinion in Colloid & Interface Science, 12,
166–178.
Tolstoguzov, V. B. (1991). Functional properties of food proteins and role of
protein-polysaccharide interaction. Food Hydrocolloids, 4, 429–468.
https://doi.org/10.1016/S0268-005X(09)80196-3.
Tolstoguzov, V. B. (2007). Ingredients interactions in complex foods:
Aggregation and phase separation. In D. J. McClements (Ed.), Understanding
and controlling the microstructure of complex foods (pp. 185–206). Cambridge:
Wood Head Publishing.
Urista, M. C., Álvarez, F. R., Riera, R. F., Cuenca, A. A., & Téllez, J. A.
(2011). Review: Production and functionality of active peptides from milk.
Food Science and Technology, 4, 293–317.
https://doi.org/10.1177/1082013211398801.
Vidigal, M. C. T. R., Minim, V. P. R., Ramos, A. M., Ceresino, E. B.,
Diniz, M. D. M. S., Camilloto, G. P., & Minim, L. A. (2012). Effect of
whey protein concentrate on texture of fat-free desserts: Sensory and
instrumental measurements. Food Science and Technology, 32, 412–418.
https://doi.org/10.1590/S0101-20612012005000047.
Voswinkel, L., & Kulozik, U. (2014). Fractionation of all major and minor
whey proteins with radial flow membrane adsorption chromatography at lab
and pilot scale. International Dairy Journal, 39, 209–214.
https://doi.org/10.1016/j.idairyj.2014.06.012.
Walstra, P., Wouters, J. T. M., & Geurts, T. J. (2006). Dairy science and
technology (2nd ed). Taylor & Francis Group; New York, EUA
14 Comprehensive Reviews in Food Science and Food Safety r Vol. 0, 2018
Wally, J., & Buchnan, S. K. (2007). A structural comparison of human serum
transferrin and human lactoferrin. Biometals, 20, 249–262.
https://doi.org/10.1007/s10534-006-9062-7.
Walzem, R. L., Dillard, C. J., & German, J. B. (2002). Whey components:
Millennia of evolution create functionalities for mammalian nutrition: What
we know and what we may be overlooking. Critical Reviews in Food Science
and Nutrition, 42, 353–375. https://doi.org/10.1080/10408690290825574.
Wang, X., Wang, Y. W., Ruengruglikit, C., & Huang, Q. (2007). Effects of
salt concentration on formation and dissociation of β-lactoglobulin/pectin
complexes. Journal of Agricultural and Food Chemistry, 55, 10432–10436.
https://doi.org/10.1021/jf071787g.
Wang, Q., Lei, H., Jiang, L., Fu, J., Liu, Y., Wen, Q., & Zhong, Y. (2014).
Optimization and evaluation of microencapsulation of star anise oleoresin.
Journal of Food Processing and Preservation, 38, 2129–2136.
https://doi.org/10.1111/jfpp.12193.
Wang, B., Blanch, E., Barro, C. J., & Adhikari, B. (2017). Preparation and
study of digestion behavior of lactoferrin-sodium alginate complex
coacervates. Journal of Functional Foods, 37, 97–106.
https://doi.org/10.1016/j.jff.2017.07.044.
Wee, M. S. M., Nurhazwani, S., Tan, K. W. J., Goh, K. K. T., Sims, I. M., &
Matia-Merino, L. (2014). Complex coacervation of an
arabinogalactan-protein extracted from the Meryta sinclarii tree (puka gum)
and whey protein isolate. Food Hydrocolloids, 42, 130–138.
https://doi.org/10.1016/j.foodhyd.2014.03.005.
Weinbreck, F. C. J., Kruif, C. G., & Schrooyen, P. (2003a). Complex coacervates
containing whey proteins. Patents WO03106014 and EP1371410.
Weinbreck, F., de Vries, R., Schrooyen, P., & de Kruif, C. G. (2003b).
Complex coacervation of whey proteins and gum arabic. Biomacromolecules,
4, 293–303. https://doi.org/10.1021/bm025667n.
Weinbreck, F., Tromp, R. H., & de Kruif, C. G. (2004a). Composition and
structure of whey protein/gum arabic coacervates. Biomacromolecules, 5,
1437–1445. https://doi.org/10.1021/bm049970v.
Weinbreck, F., Minor, M., & de Kruif, C. G. (2004b). Microencapsulation of
oils using whey protein/gum arabic coacervates. Journal of Microencapsulation,
21, 667–679. https://doi.org/10.1080/02652040400008499.
Xu, A. Y., Melton, L. D., Jameson, G. B., Williams, M. A. K., &
McGillivray, D. J. (2015). Structural mechanism of complex assemblies:
Characterisation of beta-lactoglobulin and pectin interactions. Soft Matter,
11, 6790–6799. https://doi.org/10.1039/C5SM01378J.
Yada, R. Y. (2004). Protein in food processing (1st ed). New York: CRC Press.
Yan, Y., Kizilay, E., Seeman, D., Flanagan, S., Dubin, P. L., Bovetto, L., . . .
Schmitt, C. (2013). Heteroprotein complex coacervation: Bovine
β-lactoglobulin and lactoferrin. Langmuir, 29, 15614–15623.
https://doi.org/10.1021/la4027464.
Zeeb, B., Mi-Yeon, L., Gibis, M., & Weiss, J. (2018). Growth phenomena in
biopolymer complexes composed of heated WPI and pectin. Food
Hydrocolloids, 74, 53–61. https://doi.org/10.1016/j.foodhyd.2017.07.026.
Zhang, Z. Q., Pan, C. H., & Chung, D. (2011). Tannic acid cross-linked
gelatin–gum arabic coacervate microspheres for sustained release of allyl
isothiocyanate: Characterization and in vitro release study. Food Research
International, 44, 1000–1007.
https://doi.org/10.1016/j.foodres.2011.02.044.
Zhang, N., & Mutilangi, W. (2013). Complex coacervates, methods and food
products. US patent 20130004640 A1.
Zhang, S., Zhong, Z., & Vardhanabhuti, B. (2014). Effect of charge density
of polysaccharides on self-assembled intragastric gelation of whey
protein/polysaccharide under simulated gastric conditions. Food & Function,
5, 1829–1838. http://10.1039/C4FO00019F.
Zuvanov, V. C., Garcia-Rojas, E. E., Souza, C. J., de Gulão, E. S., & Pereira,
L. J. B. (2014). Obtaining process of interpolymeric complexes from
lactalbumin, xanthan gum and pectin. Brazilian Journal of Food Technology,
17, 213–220. https://doi.org/10.1590/1981-6723.0814.

C 2018 Institute of Food Technologists®