LWT - Food Science and Technology 90 (2018) 254–264

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LWT - Food Science and Technology

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Complex coacervation: Encapsulation and controlled release of active agents T

in food systems
Noushin Eghbala, Ruplal Choudharyb,∗
a
Department of Food Science, Engineering and Technology, University College of Agriculture and Natural Resources, University of Tehran, PO Box 4111, Karaj 31587-
77871, Iran
b
Department of Plant, Soil and Agricultural Systems, Southern Illinois University, Carbondale, IL 62901, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Complex coacervation is a liquid-liquid phase separation phenomenon occurring between oppositely charged
Complex coacervation biopolymers through electrostatic interaction. Coacervates are generally produced from proteins and poly-
Microencapsulation saccharides thus they can have a wide range of functionality in various food products. Different factors such as
Rheological property pH, polymer concentration, polymer mixing ratio, ionic strength and thermal treatments have significant effect
Controlled release
on the formation of complex coacervates. Practical application of coacervates depends on their viscoelastic
Food systems
properties which has been discussed in this review. Microencapsulation by complex coacervation method is
increasingly being utilized in food industry as a result of high encapsulation efficiency and mild processing
conditions. This review focused on discussing the most recent studies on the optimization of complex coa-
cervation of different charged biopolymers, microencapsulation of various compounds by complex coacervation
and controlled release of agents in food and edible ingredients from coacervates.

1. Introduction hydrophobic interactions establishing during coacervation process

The separation of colloidal systems into two liquid phases is known
as coacervation. Coacervate refers to the phase which is more con-
centrated in component and equilibrium solution refers to other phase
(de Kruif, Weinbreck, & de Vries, 2004). Coacervates are made up of
oppositely charged species like colloids, proteins and surfactants which
are assembling in an aqueous solution. Two phases including polymer-
poor and polymer rich phases are produced upon liquid-liquid phase
separation (Singh et al., 2016). It is possible to bring a macromolecular
solute to coacervation phase separation through mixing various ratios
of water and alcohols for the coacervation equilibrium. Separation of
water soluble macromolecule into two phases at a special water/alcohol
ratio results in formation of one phase with a high concentration of
macromolecule in equilibrium with second phase containing a higher
water concentration. This phenomenon is known as “simple coacerva-
tion”. But the term complex coacervation refers to the system having
two charged macromolecules in a single solvent demixing into two or
more phases that each phase have both polymers (Veis, 2011). Complex
coacervation usually occurs when electrostatic interactions are estab-
lished between oppositely charged biopolymers in aqueous media and
there is an electrostatic equilibrium in the concentrated phase. How-
ever, less is known about secondary hydrogen bonding and
which are most likely both biopolymer and solvent dependent (Liu,
Shim, Wang, & Reaney, 2015:; Liu, Low, & Nickerson, 2009; Weinbreck,
de Vries, Schrooyen, & de Kruif, 2003). Tiebackx (1911) was first who
discussed about the coacervation but originally Bungenberg de Jong
and Kruyt (1929) investigated complex coacervation process in pro-
tein/polysaccharide system (gelatin/gum arabic). Overbeek and Voorn
(1957) developed a first successful theoretical model of complex coa-
cervation. In this relation, the modern theory of polymeric complex
coacervation was reviewed recently by Sing (2017).
Despite being miscible in the solvent, coacervates are formed
through the solute electrostatic interactions. However, more in-
vestigations should be carried out for better understanding of the
complex mechanisms of interactions occurring between different bio-
polymers leading to production of coacervates. Complex coacervation
can be used for encapsulation, biomimetic systems, formation of
packaging films and production of food emulsions or gels. The formed
coacervates are also used as encapsulants, additives, emulsifiers and
viscosity modifiers in food industry (Sing, 2017).
Encapsulation can be defined as a process of packaging active agents
within a carrier material to improve delivery of active compounds into
food products. Different natural food components such as enzymes,
carotenoids, vitamins, polyphenols and volatile essential oils are

∗
Corresponding author. 1205 Lincoln Drive Room 176, Carbondale, IL 62901, USA.
E-mail address: choudhry@siu.edu (R. Choudhary).

https://doi.org/10.1016/j.lwt.2017.12.036
Received 18 May 2017; Received in revised form 26 October 2017; Accepted 13 December 2017
Available online 14 December 2017
0023-6438/ © 2017 Elsevier Ltd. All rights reserved.
N. Eghbal, R. Choudhary
entrapped in biopolymer microparticles and nanoparticles for main-
taining their main characteristics unchanged. There are many reasons
for applying encapsulation in food industry: (i) protection of entrapped
active agent against nutritional value losses and interacting with
harmful environmental factors (e.g. heat, light, air and moisture); (ii)
decreasing evaporation and transfer rate of the core agent to the out-
side; (iii) physical properties of original substances can be modified like
converting liquid ones to a dry solid system for easy handling; (iv) re-
lease of core material can be controlled as delayed (time) or long-acting
(sustained) release; (v) masking unpleasant organoleptic properties like
odor and taste of some compounds; (vi) prevention of the incompat-
ibility between components of entrapped mixture; (vii) encapsulated
agent can be diluted when a very small amount is required (Narsaiah,
Jha, Wilson, Mandge, & Manikantan, 2014).
In this review, we mainly focused on the various parameters influ-
encing the formation of complex coacervates, microstructural, rheolo-
gical properties of coacervates, microencapsulation of different active
agents via complex coacervation and controlled release of active agents
from fabricated coacervates. An attempt was made to understand and
discuss the most recent research works which have been done in the
field of complex coacervation with the possible application in the food
industry.
2. Factors influencing complex coacervation
Two approaches are mostly used for the optimization of the complex
coacervation process between charged macromolecules namely, surface
charge density (zeta potential) and turbidity as a function of pH and
polymer ratio. Surface electrical properties of biopolymers is an im-
portant parameter for verifying complexation and stability of formed
coacervates (Eghbal et al., 2016; Wang, Adhikari, & Barrow, 2014).
Using UV-Vis spectrophotometer for measuring the turbidity of polymer
mixtures can also show the formation of complex coacervates. The
turbidity of clear initial solution containing one type of biopolymer
increases by addition of another biopolymer solution or by adjusting pH
due to the production of complex particles. However, reaching a pla-
teau of turbidity value may indicate electrostatically interaction of all
possible negative and positive charges of examined biopolymers re-
sulting in coalescence of all complex coacervates and formation of
visible insoluble coacervate phase (Eghbal et al., 2016). According to
Timilsena, Wang, Adhikari, and Adhikari (2017), this step of com-
plexation is called as macro-coacervation consisting of coacervate phase
(polymer-rich phase) and equilibrium phase (polymer-dilute phase).
Different parameters have significant effect on the process of complex
coacervation including material properties like biopolymer molecular
weight, total biopolymer concentration, biopolymer mixing weight
ratio, biopolymer charge density/biopolymer flexibility along with
process parameters like pH, pressure/stirring, temperature and ionic
strength as showed in Fig. 1 (Weinbreck et al., 2003).
2.1. pH, biopolymer ratio and ionic strength
Generally, gums and some of the natural polysaccharides like pectin
and alginate carry net negative charge in a wide spectrum of pH and
charge of proteins depends on the pH of solution (having positive
charge at pH value below its isoelectric point (pI)). So, pH is considered
as a significant factor for the complexation of biopolymers because the
optimum complexation can be obtained at pH value where both of the
macromolecules are oppositely charged. Binding of protein molecules
on polysaccharide chains at the first critical pH (pHc) lead to the for-
mation of the primary soluble complexes, and subsequently the formed
protein/polysaccharide complexes begin to aggregate into insoluble
complexes at the second critical pH (pHφ1). At this pH, macroscopic
changes in turbidity occurs until achieving a maximum (at pHopt or
pHmax), where neutral complexes are formed because oppositely
charged biopolymers reach an electrical equivalence (Ayree &
255
LWT - Food Science and Technology 90 (2018) 254–264
Nickerson, 2012; Liu et al., 2009). Protein/polysaccharide coacervates
can dissociate into soluble complexes reducing pH down to the third
critical pH (pHφ2) (Wang, Lee, Wang, & Huang, 2007).
Charge balance between proteins and polysaccharides depends on
the biopolymer ratio, which significantly influences the intensity of
complex formation (Ye, 2008). The most critical point in the complex
coacervation process is defining optimum conditions. Table 1 shows the
optimum conditions such as pH and biopolymer ratio for complex
coacervation of some oppositely charged biopolymers. Chang, Gupta,
Timilsena, and Adhikari (2016) studied protein to polysaccharide ratio,
pH and strength of electrostatic interactions for optimization of the
complex coacervation between canola protein isolate (CPI) and chit-
osan. CPI/chitosan ratio of 16:1 and pH range of 5.8–6.2 were found as
the optimum conditions for complex coacervation between studied
protein and polysaccharide. The thermal stability of CPI in complexed
form with chitosan was higher than free CPI shown by the higher peak
denaturation temperature (Td) and the denaturation enthalpy (ΔHd)
(Td = 92.3 °C, ΔHd = −7.1 mW/g and Td = 77 °C, ΔHd = −3.8 mW/g,
respectively). Also, crosslinking of the complex coacervates with
transglutaminase could increase the thermal stability of coacervates.
Liu et al. (2015) investigated the effects of pH (6.0–1.4), biopolymer
mixing ratio (1:15 to 15:1 w/w) and destabilizing agents (NaCl and
urea) on the formation of complex coacervates between bovine serum
albumin (BSA) and whole flaxseed gum (FG) (Linum usitatissum L). In
spite of carrying negative charges at pHc 5.4 > pI of BSA = 4.9, so-
luble complexes of two biopolymers were formed as a result of inter-
actions between positive patches of BSA and carboxyl groups of FG
molecules. Turbidity increased at pHφ1 4.8 illustrating the formation of
insoluble complexes between oppositely charged macromolecules. The
highest amount of insoluble complexes were formed at pHmax 3.4 above
which dissociation occurred due to progressive protonation of the FG
carboxyl groups. The ratio of protein to polysaccharide (R) with con-
stant total biopolymer concentration played an important role in coa-
cervation thus turbidity and zeta-potential increased with increasing of
BSA portion. R had positive correlation with pHφ1, pHmax and pHφ2 but
did not significantly affect pHc. Authors illustrated that independence
of pHc to R was observed by Vinayahan, Williams, and Phillips (2010)
for BSA/gum arabic mixtures, as soluble complexes could be formed
between a single polysaccharide and given amount of protein (∼10
BSA molecules per gum arabic molecule). Addition of NaCl to BSA/FG
media decreased pHc, pHφ1 and pHmax due to its disruptive effect on
electrostatic interactions between biopolymers. Turbidity analysis
showed that the amount of complex coacervates were lowered in the
presence of 150 mM urea which can break hydrogen bonds indicating
stabilizing effect of hydrogen bonds on formed complexes. In conclu-
sion, insoluble complexes with highest electrostatic interactions were
formed when the electrical charge of biopolymer mixtures were neu-
tralized. In consistent with results obtained by Liu et al. (2015), the
values of pHc were independent of initial protein/polysaccharide ratio
for the coacervation between whey proteins and carrageenan
(Weinbreck, Wientjes, Nieuwenhuijse, Robijn, & de Kruif, 2004). In
another study, the effect of pH (acidification with glucono-δ-lactone
(GDL)), ionic strength and protein to polysaccharide ratio on complex
coacervation of puka gum (extracted from the Meryta sinclaii tree) with
whey protein isolate was studied by Wee et al. (2014). Increase in
turbidity indicated that soluble complexes were formed at pHc 5.7
which is above the pI of whey protein (5.1) because of interactions
between positive patches of WPI and negatively charged carboxyl
groups on puka gum. Sharp increase in particle size as well as turbidity
at pHφ 4.7 explained association of soluble complexes due to presence
of more positively charged protein molecules interacting with puka
gum and neutralizing the complexes. So, as a consequence below pHφ,
zeta potential of the mixtures showed rapid increase toward neutrality.
On the other hand, increasing ratio of protein to polysaccharide re-
sulted in increasing of pHφ as a result of presence of more positively
charged protein molecules. They found that addition of high salt
N. Eghbal, R. Choudhary LWT - Food Science and Technology 90 (2018) 254–264

Fig. 1. Physicochemical factors influencing complex coacervation

process (Schmitt & Turgeon, 2011).

Table 1
Formation of complex coacervates under optimum pH and polymer mixing ratio.

Biopolymers pH range Optimum conditions References

Cationic biopolymers Anionic biopolymers pH Biopolymer ratio

PPI Gum arabic 1.5–6.0 3.5 2:1 Liu et al., 2010

LPI Gum arabic 1.5–8.0 3.5 1:1 Ayree & Nickerson, 2012
CPI Carrageenan 1.5–7.5 5.0 15:1–20:1 Stone et al., 2013
β-lg Carrageenan 0.5–7.0 1.0–2.0 2:1 Hosseini et al., 2013
WPI Puka gum 3.4–7.0 3.6 2:1 Wee et al., 2014
4:1
WPI Acacia gum 2.5–5.0 3.1–3.4 1:1 Ach et al., 2015
3.1–4.0 2:1 and 4:1
FPI FG 1.5–8.0 3.1 3:1 Kaushik, Dowling, Barrow, & Adhikar, 2015
OVA Gum arabic 1.5–6.5 3.71 2:1 Niu et al., 2015
BSA FG 1.4–6.0 3.4 2:1 Liu et al., 2015
PL Gum arabic 1.0–12.0 4.0 – Gulao et al., 2016
Chitosan CPI 4.0–8.0 6.0 1:16 Chang et al., 2016

CPI: canola protein isolate, PL: polypeptide leucine, CSG: chia seed gum, WPI: whey protein isolate, FPI: flaxseed protein isolate, FG: flaxseed gum, OVA: ovalbumin, BSA: bovine serum
albumin, β-lg: β-lactoglobulin, LPI: lentil protein isolate, PPI: pea protein isolate.

concentration (≥60 mM NaCl) contributed to absolute prevention of
complex coacervation. Also, it was reported by Wang et al. (2007) that
high concentration of salt (CNaCl > 0.21 M) significantly reduced real
β-lg/pectin ratio in coacervates. However, it is worth mentioned that
addition of low salt concentration can have positive effect on the for-
mation of protein polysaccharide complex coacervates through over-
coming short-range repulsions between macromolecules and leading to
exposure of more available sites on proteins like whey protein for in-
teractions (Schmitt, Aberkane, & Sanchez, 2009, pp. 420–476:; Wee
et al., 2014).
Recently, complex coacervation of chia seed protein isolate (CPI)
and chia seed gum (CSG) as a function of pH and protein/poly-
saccharide ratio has been reported (Timilsena, Wang, Adhikari, &
Adhikari, 2016). The highest amount of complex coacervates were
formed at pHopt 2.7 and CPI to CSG ratio of 6:1 mainly by electrostatic
interactions. Based on differential scanning calorimetry (DSC) results,
thermal stability of protein increased in coacervates and addition of
transglutaminase as a crosslinking agent had positive effect on their
thermal stability properties. Ach et al. (2015) investigated complex
coacervation of WPI with acacia gum (AG) and compared with coa-
cervation of pure β-lg/AG and α-lac/AG. Based on results obtained by
capillary gel electrophoresis for WPI/AG mixtures at different protein/
polysaccharide ratios, the concentration of β-lg and α-lac reached to
minimum at pH 3.5 relating to formation of maximum coacervates.
However, at acidic pH (< 2.3), protein concentration increased in su-
pernatant indicating dissociation of complexes. The pH, corresponding
to the formation of complex coacervates thus resulting in decreasing of
protein concentration, was R dependent. It was shown that β-lg con-
centration reduced in high extent to that of α-lac as a result of estab-
lishing stronger interactions with polysaccharide originating from its
higher molar mass and pI. Pure α-lac could form complex coacervates at
lower pH (3.4) than that of pure β-lg (pH 4.5) with AG due to more
acidic pI of α-lac. In another study, Weinbreck et al. (2003) investigated
the effect of different parameters influencing complex coacervation of
whey proteins/gum arabic. It was seen that for pH range between pHc
and pHφ1, the radius of formed complexes decreased significantly
comparing to the blank of gum arabic while scattering intensity in-
creased which can be due to increase in molar mass of particles. Pro-
duced particles within mentioned pH range had net negative charge
consequently were soluble in the solvent. However, by decreasing pH
below pHφ1, phase separation occurred as explained by high increase in
scattering intensity, turbidity and particle size. For whey protein/gum
arabic complexation, β-lg was predominant regarding pHφ1 of 4.7 for
the coacervation process and pI of β-lg 5.2 and 4.1 for α-la. Increasing
of ionic strength reduced the pH at which interactions were established
between macromolecules as a result of screened charges of polymers by
microions. A high salt concentration (> 54 mM) was defined as the
point of salt resistance which refers to the amount of salt necessary for
prevention of coacervation. They found that stable values of pHc for
different biopolymer concentrations (Cp) could illustrate the theory
that formation of soluble complexes is independent of Cp. Also, in-
creasing the total biopolymer concentrations had no significant effect

256
N. Eghbal, R. Choudhary
on the shift of pHφ1 values. Liu et al. (2010) used turbidimetric analysis
for investigating the nature of Coulombic and non-Coulombic interac-
tions of pea protein isolate (PPI)/gum arabic complexation in the pre-
sence of destabilizing agents (NaCl and urea) as a function of pH
(4.3–2.4). Addition of NaCl disrupted electrostatic interactions between
charged macromolecules consequently preventing formation of com-
plexes due to action of Na+ and Cl− ions on screening charges of
macromolecules. But for urea, critical pH values (pHc 4.2, pHφ1 3.7 and
pHopt 3.6) decreased to lower values, which is known that urea disturbs
both hydrogen and hydrophobic bonds of formed complexes.
The effect of pH and biopolymer mixing ratio on the formation of
soluble and insoluble canola protein isolate (CPI)/carrageenan (CG)
complexes as well as their functional properties compared to protein
were studied by Stone, Cheung, Chang, and Nickerson (2013). CPI/CPI
aggregations were suppressed by addition of CG as shown by decreased
optical density (OD) of mixed solution due to the presence of strong
electrostatic forces induced by the sulfate groups of free CG molecules.
According to electrophoretic mobility measurements, isoelectric point
of CPI was 5.78 at which OD reached to its peak and by addition of
polysaccharide the net neutrality pH of protein was shifted to more
acidic pH values. It was observed that increasing mixing ratios resulted
in formation of soluble and insoluble complexes at pH values above
protein pI where negatively charged CG molecules interacted with
positively charged patches on protein surface. Solubility, foaming ca-
pacity and emulsion capacity of protein decreased significantly through
complexation with CG at two pH values of 6.7 and 5.0 corresponding to
formation of soluble and insoluble complexes, respectively. But addi-
tion of polysaccharide had no negative effect or even increased foaming
stability and emulsion stability of the CPI alone.
The influence of pH, protein/polysaccharide ratio and total biopo-
lymer concentrations on flaxseed protein isolate (FPI)/flaxseed gum
(FG) complexation along with structural changes of protein upon acidic
titration were determined by Kaushik, Dowling, Barrow, and Adhikari
(2015). It was observed that secondary structure of flaxseed protein
underwent a significant change through decreasing pH from 8.0 to 3.0
as β-sheets and random coils were formed at high rate while helix
structure were dissociated. Turbidity values of mixed solutions were
higher than protein alone indicating formation of complexes which
started at pHc 6.0 (above pI of protein 4.2). According to the results,
increasing protein/polysaccharide ratio from 1:1 to 15:1 increased OD
values. However, there was no significant difference between OD values
of protein/polysaccharide from 3:1 to 15:1 illustrating complexation of
all FG negative charges with FP positive sites at optimum rate of 3:1.
Despite carrying negative charges, insoluble complex coacervates were
formed at pHφ1 4.5 and complexation between oppositely charged
macromolecules increased at lower pH values than protein pI thus
reached to maximum at pHopt 3.1. The effect of pH and FPI to FG ratio
on complex coacervation was exhibited by phase diagram indicating
dependency of pH1 and pHopt to the mixing ratios. Ayree and Nickerson
(2012) studied the effect of pH and biopolymer mixing ratio on the
formation of soluble and insoluble lentil protein isolates (LPI)/gum
arabic complexes. Formation of soluble complexes between LPI and
gum arabic occurred at pH 5.8 as OD increased slightly and insoluble
complexes were formed at pH 3.6 confirmed by sharp increase of tur-
bidity value. Due to protonation of carboxyl functional groups on gum
arabic backbone at low pH values (pH < pHopt), formed complexes
began to dissociate resulted in reduction of OD value. Gum arabic
molecules interacted with all positive charges of lentil proteins at the
mixing ratio of 1:1 LPI/gum arabic (zeta potential: 0 mV) above which
excess amount of protein in the solution contributed to LPI/LPI ag-
gregation rather than complexation. It was seen that complexation with
dehulled lentil protein turned the critical pH values to higher ones
which can be related to both presence of hydrophobic interactions and
removal of polyphenols, tannins and fiber from the flour during de-
hulling.
Niu et al. (2015) investigated the effect of mixing ratios and salt on
257
LWT - Food Science and Technology 90 (2018) 254–264
ovalbumin (OVA)/gum arabic coacervation as well as OVA structural
changes by spectroscopic techniques. Decreasing pH from pHc 4.6 to
pHφ1 4.2, increased the turbidity value and charge neutralization oc-
curred at pH 3.7. Macromolecules carried net opposite charges in the
pH range of 2.5–4.0 thus strong electrostatic interactions established
resulted in sharp increase of mean particle diameter at pH 4.0 that
decreased above and below mentioned pH range. Analysis of mean
particle diameter versus time showed that size of complexes increased
rapidly during first 12 min at pH 3.7 indicating importance of the
strength of the electrostatic interactions between OVA/gum arabic on
phase separation. Increasing protein/polysaccharide ratio from 1:1 to
24:1 shifted turbidity curve to higher pH values and turbidity value
reached maximum for 2:1 mixing ratio which corresponded the highest
amount of coacervate formation. It was observed that addition of MgCl2
and BaCl2 downshifted pHopt and reduced coacervation yield more than
NaCl due to incorporation of divalent cations in the complexes and
neutralization by gum arabic reactive groups. According to results ob-
tained by second-derivative UV spectrum, addition of polysaccharide to
OVA solution unfolded its tertiary structure that at pH 4.0 more tyr-
osine residues were at the exposer of solvent. Fluorescence intensity of
biopolymer mixtures decreased and showed blue shift confirming
changes in tertiary structure of protein resulted in sheltering some
tryptophan residues in hydrophobic environment during structure un-
folding. Burova et al. (2007) studied the influence of complexation with
bovine β-casein on conformational changes of ι and κ-carrageenans. The
highest rate of protein aggregation was obtained at about pH 4.2 while
the light scattering intensity of protein solution decreased upon addi-
tion of polysaccharide as a result of enlarging the solubility pH range of
protein lower region. Complexation with β-casein resulted in carra-
geenans conformational changes by reducing the helicity of carragee-
nans as function of pH and increased content of protein in formed
complexes. At the same ionic strength (0.15 M) ι-carrageenan had
higher binding constant than κ-carrageenan because of containing
higher charge density illustrating electrostatic nature of complex for-
mation. However, κ-carrageenan showed higher binding constant as the
ionic strength decreased (from 0.15 M to 0.03 M) indicating that release
of counterions during protein/polysaccharide complexation is the main
driving force.
Recently, Gulao, de Souza, Andrade, and Garcia-Rojas (2016) in-
vestigated the effect of pH, polysaccharide concentrations and ionic
strength on the formation of polypeptide leucine (PL) and gum arabic
complex coacervates. The results showed that the highest amount of
complex coacervates with largest particle size were formed at 0.03%
gum arabic concentration at which the ζ-potential value was close to
zero. Increasing polysaccharide concentration decreased particle size
indicating dissociation of insoluble complexes and formation of soluble
complexes. The electrostatic interactions between biopolymers estab-
lished at pHφ1 4.0 (close to pI of polypeptide) which was confirmed by
increase in turbidity and particle size. At pHφ2 2.0, which is the pKa of
gum arabic, turbidity decreased significantly due to the electrostatic
repulsion and formation of soluble complexes. At the same biopolymer
concentration, the addition of salt (NaCl) resulted in decreasing of
turbidity as well as particle size. The morphology studies of formed
complex coacervates by X-ray diffraction exhibited crystalline regions
which have lower solubility compared to amorphous structure.
2.2. Thermal treatment
The effect of thermal treatment and pH on the properties of soy
protein isolate-pectin complexes was investigated by Jaramillo,
Roberts, and Coupland (2011). Formation of complexes occurred at pH
3.0 (below protein pI (pH 4.0 and 5.0)) which was confirmed by de-
creased ζ-potential value of protein solution upon addition of poly-
saccharide. Solubility of protein moderately decreased in the presence
of pectin at higher pH values (6.0–7.0) because of non-adsorbed pectin
which led to depletion interaction between proteins. Applying thermal
N. Eghbal, R. Choudhary
treatment (30 min, 90 °C) to formed SPI/pectin complexes increased
solubility around pI of protein while decreased at pH 3.0 compared
with unchanged profile of protein solubility upon raising temperature.
Authors concluded that SPI/pectin complexes are heat resistant and
underwent structural modifications through thermal treatment which
affect their solubility. Souza, Garcia Rojas, Melo, and Lins (2013),
studied the effect of temperature, pH, and polysaccharide types (pectin,
xanthan gum, carrageenan and carboxymethylcellulose (CMC)) on the
formation of egg yolk lipoprotein complex coacervates. Regarding pI of
yolk protein (4.5 or 4.7), turbidity values for all three studied masses
(0.025, 0.033 and 0.050 g) were low in pH range of 3.0–4.0. Increasing
of temperature and/or pH resulted in slight increase in turbidity of
mixtures from pH 6.5 and temperature 30 °C. Increasing in turbidity
was attributed to two main reasons. As temperature increases, hydro-
phobic interactions are reinforced by weakening hydrogen bonds con-
tributing to the increase of entropy. Secondly, electrostatic repulsion
becomes strong between negatively charged macromolecules away
from their pI and pKa through increasing pH. Lower pKa value of car-
rageenan (2.0) compared to pKa values of xanthan gum (2.9), CMC
(4.3) and pectin (3.5) resulted in establishing high electrostatic inter-
actions between macromolecules due to containing high proportion of
negative charges within mentioned pH range.
An attempt was made by Ayree and Nickerson (2012) to understand
the nature of 1:1 LPI (with hull)/gum arabic interactions during a pH
acid titration at room temperature and increased temperature (60 °C).
Elevating temperature downshifted critical pH values (pHφ1, pHopt,
pHφ2) also decreased turbidity value at pHopt illustrating important role
of hydrogen bonding in formation and stability of complexes. It is
known that elevating temperature disrupts hydrogen bonds and re-
inforces hydrophobic interactions between charged biopolymers. Si-
milar results were obtained by Liu et al. (2010) for studying the nature
of interactions between PPI and gum arabic biopolymers at different
temperature (6–60 °C). Increasing temperature downshifted turbidity
profile and critical pH values because of reduced hydrogen bonding
which destabilized the complexes. But turbidity and critical pH values
upshifted by decreasing temperature from 23 to 6 °C as a result of en-
hanced complex stability through increased hydrogen bonding.
2.3. Polysaccharide depolymerization
The effect of polysaccharide depolymerization through high in-
tensity ultrasound on complex coacervation between k-carrageenan
(KC) and β-lactoglobulin (β-lg) was studied by Hosseini et al. (2013).
Increasing time and amplitude of the ultrasonication process decreased
the viscosity of polysaccharide solution by producing smaller poly-
saccharide chains. As a result, solutions of sonicated k-carrageenan and
protein complexes exhibited lower turbidity than untreated ones. The
affinity constant between protein/polysaccharide was significantly re-
duced at pH 4.2 by the sonication according to isothermal titration
calorimetry (ITC) results. Sonochemical interactions decreased the
charge density of ultrasonicated (US) KC which was observed by lower
zeta potential value of (US) KC/β-lg nanoparticles than intact KC/β-lg
nanoparticles. Also, lower turbidity value of (US) KC/β-lg solutions
than intact ones was related to the formation of smaller polysaccharide
chains after sonication. Sonication had positive effect on the homo-
genization of nanoparticles present in the mixture. Soluble complexes
were formed at pHc (∼5.3–5.5) above pI of β-lg (∼4.7–5.2) and critical
pHφ1 (∼4.8) was reached at lower pH values due to nucleation and
growth. The highest amount of interactions between β-lg and KC were
established at pH 1.0–2.0 according to obtained maximum optical
density. The dissociation of complexes did not happen even at lower pH
values. ITC measurements showed exothermic sequence for interactions
of β-lg with KC which is associated with electrostatic neutralization of
biopolymers.
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3. Microstructural and rheological properties of coacervates
One of the crucial parameters in practical application of coacervates
is their viscoelastic property. Rheological properties of various complex
coacervates formed by fish gelatin/gum arabic (Anvari, Pan, Yoon, &
Chung, 2015), gum arabic/chitosan (Espinosa-Andrews, Sandoval-
Castilla, Vazequez-Torres, Vernon-Carter, & Lobato-Calleros, 2010),
agar/gelatin (Singh, Aswal, & Bohidar, 2007), β-lactoglobulin/pectin
(Wang et al., 2007), BSA/pectin (Ru, Wang, Lee, Ding, & Huang, 2012)
and peptide leucin/gum arabic (Gulao et al., 2016) have been in-
vestigated. It has been reported that viscoelasticity of phase separated
complexes has positive correlation with the strength of the electrostatic
interaction between biopolymers like coacervates obtained by whey
protein/gum arabic (Weinbreck et al., 2004), chitosan/gum arabic
(Espinosa-Andrews et al., 2010) and sodium caseinate/gum tragacanth
systems (Gorji, Gorji, Mohammadifar, & Zargaraan, 2014). In this case,
complex coacervates produced by the strongest electrostatic interaction
exhibited the highest storage (elastic) (G′) and loss (viscous) modulus
(G″). In conclusion, the parameters that have significant effect on the
strength of electrostatic interactions between charged biopolymers in-
cluding pH, biopolymer ratio, phase separation temperature and ionic
strength affect the rheological properties of coacervates. The viscosity
and viscoelastic modulus of chitosan/soy protein isolate (SPI)/coa-
cervates with three mixing ratios of 0.067, 0.125 and 0.2 g/g were
evaluated at pH 6.0. The coacervates produced at mixing ratio of
0.125 g/g showed the highest viscosity due to the establishment of the
stronger electrostatic interactions between SPI/chitosan. Strain sweep
experiments were utilized for the measurement of viscoelastic modulus.
A typical shear thinning behavior of a pseudoplastic fluid was observed
for SPI/chitosan coacervates. G′ value of three mixing ratios was higher
than G″ value for the all frequency range with the maximum values
obtained for the coacervates formed at polysaccharide/protein mixing
ratio of 0.125 g/g. Like the viscosity, the highest viscoelastic modulus
were achieved for coacervates prepared at ratio where charge neu-
tralization happened (Yuan, Kong, Sun, Zeng, & Yang, 2017). However,
according to Huang, Duo, Xiao, & Wang (2017) the viscoelasticity of
complex coacervates (N, O-carboxymethyl chitosan (NOCC)/gum
arabic) does not only depend on the strength of electrostatic interaction
determined by coacervate yield. Coacervates of NOCC/gum arabic
formed at pH 6.0 showed the highest modulus values than those ob-
tained at pH 3.0 with the strongest interactions.
Anvari et al. (2015) studied the effect of phase separation tem-
perature on structural and rheological properties of fish gelatin/gum
arabic complex coacervates. Volume fraction of coacervate phase and
total biopolymer concentration, especially gum arabic molecules in the
coacervate phase increased by reducing phase separation temperature.
Fourier transform infrared (FTIR) spectroscopy showed that at 10 °C
high partition of biopolymers in coacervate phase can be related to
establishment of stronger hydrogen bonds in fish gelatin/gum arabic
coacervate phase. The coacervate phase exhibited more shear-thinning
behavior as the temperature decreased. At the same time gum arabic
concentration increased which is known that polysaccharide relaxation
have significant effect on shear-thinning behavior. Compared to bio-
polymers alone, complexes were at the exposure of structural and
conformational changes especially at lower temperatures at which hy-
drogen bonds become stronger. Contrary to high temperatures (40 and
30 °C), the storage modulus of coacervates (G′) at 10 °C increased at any
tested frequency without decreasing due to reduced molecular mobility,
increased structuration, reinforced hydrogen bonds along with in-
creased polysaccharide concentration. Liquid-like viscous behavior of
the coacervate phases was verified by the higher G″ values than G′
values at all studied temperatures.
Microstructure and rheological properties of the gum arabic/chit-
osan complex coacervates were determined by Espinosa-Andrews et al.
(2010). Regardless of pH, the loss modulus (G″) of all coacervates was
higher than storage modulus (G′) over the whole frequency range
N. Eghbal, R. Choudhary
Fig. 2. Principle of complex coacervation process (Madene, Jacquot, Scher, & Desobry, 2006).
showing liquid viscoelastic behavior. However, gum arabic/chitosan
complexes exhibited the highest viscoelastic property at pH 4.5 due to
charge balance between two macromolecules. According to SEM mi-
crographs, produced coacervates had a sponge-like microstructure with
homogeneously distributed small size agglomerated particles and het-
erogeneous sized vacuoles which interspaced the formed structure. It
was obvious that pH had significant effect on microstructure of coa-
cervates that smaller coacervates were formed at pH 4.5 than 3.0 and
6.0. Increased relative density of the coacervates at pH 4.5 resulted in
reduction of the number of the vacuoles and increasing their viscoe-
lastic moduli values. Singh et al. (2007) reported that agar/gelatin
coacervates showed non-newtonian and shear thinning behavior. It is
clear that different parameters such as pH, ionic strength, temperature
and the amount of polyanions and cations have significant effect on the
visco-elastic nature of the coacervates. Storage modulus G′(w) of agar/
gelatin coacervates was significantly higher than their loss modulus G″
(w) indicating viscous nature of formed coacervates. In another study,
the effect of salt concentration and initial protein/polysaccharide ratio
on the rheological properties of β-lactoglobulin and pectin coacervates
were studied by Wang et al. (2007). Based on dynamic rheological
measurements, coacervates (formed at protein/polysaccharide ratio of
5:1 and CNaCl 0.02 M) showed shear thinning behavior as the complex
viscosity reduced linearly with frequency. The storage modulus (G′) of
coacervates were significantly higher than the loss modulus (G″), il-
lustrating interconnected gel-like structure of coacervates. Β-lactoglo-
bulin/pectin coacervates with stronger structure and higher G′ values
formed at increased salt concentration from 0.01 to 0.21 M. It is worth
mentioned that self-aggregation of β-lactoglobulin enhanced at higher
salt concentrations (from 0.21 to 0.41) which reduced interactions of
protein with polysaccharide resulted in formation of coacervates with
loose structure and low G′ values. They found that increasing protein/
polysaccharide ratio enhanced the amount of pectin in formed coa-
cervates leading to the production of more compact coacervates with
higher elasticity. Also, Ru et al. (2012) studied the effect of added salt
concentrations and the initial protein/polysaccharide ratio on the
rheological properties of BSA/pectin coacervates. In consistent with the
results obtained by Wang et al. (2007), BSA/pectin coacervates had gel-
like network structure with G′ values higher than G″ values, exhibiting
shear thinning behavior. The G′ values of the formed coacervates had
negative correlation with added different salt concentrations. In-
creasing salt concentration resulted in reduction of biopolymers content
in coacervates forming watery structure. According to the results, the
coacervates of protein/polysaccharide can be known as a
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LWT - Food Science and Technology 90 (2018) 254–264
polysaccharide chain network which is produced from the aggregation
of protein/polysaccharide complexes while linkers are bound proteins.
So, increasing protein/polysaccharide ratio contributed to an increase
in protein molecules bound to polysaccharide chains and reaching to
charge neutrality which led to formation of coacervates with higher G′
values.
It was seen that PL (0.2%)/gum arabic (0.03%) complex coacervates
showed viscoelastic behavior at low frequencies. The values of G′ in-
creased independently of G″ values as frequency increased, verifying
complexation of PL and gum arabic with electrostatic interactions
(Gulao et al., 2016). Elastic behavior of PL/gum arabic relates the
electrostatic interactions between the peptide and polysaccharide
chain.
4. Encapsulation by complex coacervation
Entrapment of small solid particles, liquid droplets or gases inside a
thin layer of coating material (shell) is known as microencapsulation.
Generally, polymers like proteins and polysaccharides (combined or
alone) are used in microencapsulation of food ingredients. Successful
encapsulating process depends on the stability of active agents (core),
the properties of coating materials as well as suitability of the delivery
system (microcapsules) for its application (Nazzaro, Orlando, Fratianni,
& Coppola, 2012). Encapsulation of enzymes, essential oils, poly-
phenols, antimicrobial peptides and bacteria can overcome the dis-
advantages of direct addition of such agents into food matrix by con-
trolling release of agents, increasing shelf life, inhibiting partial
inactivation upon interacting with different components. Coacervation
encapsulation (simple and complex) is the phase separation of one or
many hydrocolloids from the initial solution with prepared coacervates
as a layer and suspended or emulsified active agents as the core (Fig. 2).
Microencapsulation process by complex coacervation involves four
main steps of emulsification, coacervation, gelation and hardening
(Dong et al., 2008). The structure of coacervate microcapsules depends
on the homogenization rate during emulsification process. Mono-
nuclear microcapsules are formed at low homogenization rate and
multinuclear microcapsules are prepared at high homogenization rate
(Dong et al., 2011). It is worth mentioned that the size and morphology
of prepared coacervate microcapsules significantly depend on the pro-
cessing conditions (Lemetter, Meeuse, & Zuidam, 2009). Also, nano-
particles with a lipid core can be produced by complex coacervation
process (Aloys et al., 2016).
High oxidative stability was achieved for yogurts formulated with
N. Eghbal, R. Choudhary LWT - Food Science and Technology 90 (2018) 254–264

gelatin/gum arabic coacervates modified by Maillard reaction con-
taining stearidonic acid soybean oil during 14 days of storage at 4 °C
illustrating preservation of its antioxidant properties (Ifeduba & Akoh,
2015). Applying mild heating, desolvation or crosslinking might be
needed with the purpose of increasing the mechanical and thermal
stability of the formed microcapsules (Timilsena et al., 2016). The best
method for the encapsulation of heat sensitive compounds is complex
coacervation as the thermal treatment is not applied during the process.
Crosslinking of the complex coacervates can be achieved by using
glutaraldehyde, tannic acid, gallic acid and transglutaminase. But for
the case of food application, glutaraldehyde is not appropriate choice
because of its toxic nature. The main advantages of microcapsules ob-
tained by coacervation technology are high encapsulation efficiency
(up to 99%) and the excellent controlled release properties based on
mechanical stress, temperature or sustained release (Gouin, 2004). It is
known that complex coacervation is used mainly for encapsulation of
hydrophobic compounds and contains some limitations for en-
capsulating hydrophilic substances. Applying some modifications in the
method like adding double emulsion step at the beginning of process
can lead to encapsulation of hydrophilic compounds (Mendanha et al.,
2009). Complex coacervation has been used for microencapsulation of
various active agents shown in Table 2.
Algal oil is rich in polyunsaturated fatty acids making it sensitive
against oxygen and temperature. Yuan et al. (2017) studied micro-
encapsulation of algal oil by soy protein isolate (SPI)/chitosan complex
coacervation. The highest coacervation was obtained at pH 6.0 and
chitosan/SPI ratio of 0.125 g/g according to the ζ-potential and coa-
cervation yield results. The microencapsulation of algal oil within SPI/
chitosan complex coacervates contributed to the higher encapsulation
efficiency (97.31 ± 1.16% with added transglutaminase) and oxida-
tive stability than encapsulation by SPI alone (75.4 ± 2.8%). There
was no significant difference between oxidative stability of micro-
capsules containing algal oil prepared from different protein/poly-
saccharide ratios but crosslinked microcapsules with TG displayed the
lowest hydroperoxides during storage. Monitoring the lipid hydroper-
oxides (primary oxidation product) and volatile hexanal (secondary
oxidation product) during storage with thermal acceleration were the
parameters for investigating the oxidative stability of algal oil micro-
capsules. Hardening of microcapsules by transglutaminase improved
the oxidative stability of SPI/chitosan microcapsules. Antioxidant
properties of chitosan along with the enhancement of protection against
oxidation through creation of oxygen barrier after complex coacerva-
tion and crosslinking are the main reasons for increased oxidative sta-
bility of microcapsules. Recently, the effect of matrix-wall systems
(collagen/alginate and collagen/pectin), drying methods (freeze and
spray drying) and core dispersion systems (water in oil emulsion or in
suspension) were investigated for microencapsulation of nisin with an
avocado antioxidant extract by complex coacervation by Calderón-
Oliver, Pedroza-Islas, Escalona-Buendía, Pedraza-Chaverri, and Ponce-
Alquicira (2017). Combining spray drying with emulsified core led to
higher encapsulation efficiency and yield indicating the importance of
drying method and the core incorporation. Collagen/alginate with ratio
of 1:1 showed the highest encapsulation efficiency 82.14 ± 1.18 and
81.36 ± 1.27% for nisin and avocado extract respectively at pH 3.0
(combined emulsified core with spray drying method). Collagen/pectin
with ratio of 4:1 showed the highest encapsulation efficiency
84.66 ± 1.20 and 82.96 ± 1.25% for nisin and avocado extract re-
spectively at pH 3.0 (combined emulsified core with spray drying
method). Application of non-emulsified core incorporation along with
freeze drying method resulted in the highest loading values of the two
used components. However, lower loading values of active agents can
lead to more protection as the degradation and release of agent to the
environment is fast for the case of higher loading values because the
core material is near to the surface of capsule (Oxley, 2014). Despite
having various health-promoting effect, astaxanthin is used as a food
colorant (owning intense red color). Oxidation reactions with the pos-
sibility of increasing during thermal treatment and storage can lead to
the degradation of astaxanthin. For eliminating mentioned problems,
astaxanthin-containing lipid extracts from shrimp waste was en-
capsulated through complex coacervation of gelatin/cashew gum and
the microcapsules were freeze dried. Polymer concentration, ratio, pH
and ionic strength were investigated as the parameters influencing
coacervation of gelatin/cashew gum. Biopolymers could produce
complex coacervates at pH range of 4.0–4.5. The optimum conditions
for complex coacervation were found at protein/polysaccharide ratio of
1:2.5 and pH 4.1. The encapsulation efficiency of astaxanthin was
59.9 ± 0.01% and the stability of lipid extract was enhanced by en-
capsulation (43 days/36 ± 1 °C/80% relative humidity). Encapsulated
astaxanthin resulted in the improvement in coloring capacity in yoghurt
without any changes in odor of the food compared with non-en-
capsulated agent (Gomez-Estaca, Comunian, Montero, Ferro-Furtado, &
Favaro-Trindade, 2016).
Sensitive probiotic cultures can be protected by encapsulation
method consequently resulting in improving their stability and viability
in food products. Stability of encapsulated lactic acid bacteria (LAB)
have been shown to increase in dairy products during storage time
(Aloys et al., 2016). In this relation, Eratte et al. (2015) have co-en-
capsulated tuna oil (rich in Omega-3 fatty acids) and Lactobacillus casei

Table 2
Microencapsulation of various active compounds via complex coacervation.

Biopolymers Active agent EE (%) Polymer ratio Opt pH Drying method References

SPI/pectin Casein hydrosylate 91.62 ± 0.63 1:1 4.4 Freeze drying Mendanha et al., 2009
SPI/gum arabic Sweet orange oil ∼80 1:1 4.0 Spray drying Jun-xia et al., 2011
SPI/pectin Propolis 66–72 – 4.0 Lyophilization Nori et al., 2011
Gelatin/pectin Lycopene 93.2 ± 2.1 1:1 3.0 Freeze drying Silva et al., 2012
Gelatin/gum arabic Ascorbic acid ∼98 1:1 4.4 Freeze drying Comunian et al., 2013
Gelatin/gum arabic Aspartame 45–71 1:1 4.0 Freeze drying Rocha-Selmi et al.,
2013
Gum arabic/ Miglyol – 0.25 3.6 – Butstraen & Salaün,
chitosan 2014
WPI/gum arabic Tuna oil and probiotic Lactobacillus 93.35 ± 3.01 for SD WPI-P-O-GA – 3.75 Spray drying and freeze Eratte et al., 2015
casei 431 drying
Gelatin/cashew gum Astaxanthin 59.9 ± 0.01% 1:2.5 4.1 Freeze drying Gomez-Estaca et al.,
2016
Collagen/alginate Nisin (n) and an avocado 82.14 ± 1.18(n) 1:1 3.0 Spray drying and freeze Colderon-Oliver et al.,
Collagen/pectin Antioxidant extract (a) 81.36 ± 1.27(a) 1:1 3.0 drying 2017
84.66 ± 1.20(n)
82.96 ± 1.25(a)
Chitosan/SPI Algal oil 97.31 ± 1.16 0.125 g/g 6.0 – Yuan et al., 2017

SPI: soy protein isolate, WPI: whey protein isolate, SD: spray dried, P: probiotic, O: oil.

260
N. Eghbal, R. Choudhary
431 in whey protein isolate (WPI)/gum arabic complex coacervates
which turned into powder by spray drying and freeze drying. According
to the results, the viability of probiotic cells (P) improved in the pre-
sence of oil compared with WPI/P/gum arabic microcapsules. Also,
drying method had significant effect on viability of bacteria. Due to
higher inlet and outlet temperatures as well as stresses induced by
thermal and dehydration steps, spray drying contributed to lower via-
bility of L. casei cells compared with freeze drying. On the other hand,
the authors found that oxidative stability of oil (O) was higher in spray
dried WPI/P/O/gum arabic microcapsules as a result of containing
lower surface oil. In another study, Mendanha et al. (2009) used fixed
ratio of soy protein isolate and pectin concentrations (1:1) adjusted at
pH 4.4 as wall materials to microencapsulate casein hydrosylate by
complex coacervation. Formed coacervate microcapsules exhibited low
solubility in water resulting in controlled release of core agent. By in-
creasing casein hydrosylate concentration, the encapsulation efficiency
decreased and solubility rate increased. It was noticed that micro-
encapsulation by coacervation reduced hygroscopicity and bitter taste
of casein hydrosylate with best results obtained for sample containing
50% core agent than 100% and 150% concentrations (encapsulation
efficiency: 91.62 ± 0.63, 87.92 ± 0.72 and 78.80 ± 90%, respec-
tively). Jun-xia, Hai-yan, and Jian (2011) microencapsulated sweet
orange oil by complex coacervation with different SPI/gum arabic ra-
tios and converted to powder by spray drying. The highest degree of
coacervation occurred at pH 4.0 which was the electrical equivalence
point (EEP) of the SPI/gum arabic system and at SPI/gum arabic ratio
1:1. It was shown that core material load had significant effect on mi-
croencapsulation yield (MEY) which decreased by increasing of core
load. Among studied micromolecules such as sucrose, maltodextrin,
polyethylene glycol (PEG) 2000 and PEG 4000 for their effect on
complex coacervation, only sucrose resulted in increasing of MEY. Both
microencapsulation efficiency and yield reached to the highest values at
sucrose/SPI ratios of 1:1. Finally, well retention of D-limonen (major
component of sweet orange oil) was an indication for its successful
encapsulation by complex coacervation. In another study, Dong et al.
(2008) studied the effect of different crosslinking parameters on pro-
duction of transglutaminase-hardened spherical multinuclear gelatin/
gum arabic microcapsules containing peppermint oil through complex
coacervation. It was shown that increasing crosslinking time (6 h) and
temperature (15 °C) contributed to increased microcapsule stability due
to enhanced intermolecular crosslink density in gelatin which led to low
oil release. The most structurally stable microcapsules were formed at
pH 6.0 at which transglutaminase showed the highest enzymatic ac-
tivity in the case of using gelatin as substrate. Also low amount of oil
released from microcapsules hardened by high concentration of trans-
glutaminase (15 U g−1 of gelatin).
Comunian et al. (2013) used double emulsion method followed by
complex coacervation with gelatin/gum arabic for microencapsulation
of ascorbic acid (AA) known as an efficient antioxidant but unstable
agent. The high encapsulation efficiency (between 97.33 ± 0.81 and
99.57 ± 0.32%) was an illustration for successfulness of applied
method in microencapsulation of AA. Low water activities below 0.6 for
all formed microcapsules indicated their microbial stability. The
highest amount of retained AA at both 20 and 37 °C was for micro-
capsules of gelatin/gum arabic/AA at ratios of 1:1:0.75 with 0.025 g/
mL polymer because of having larger mean diameters resulting in lower
exposure of AA surface. Also in another work, Rocha-Selmi, Bozza,
Thomazini, Bolini, and Favaro-Trindade (2013) used double emulsion
method followed by complex coacervation for microencapsulation of
aspartame with gelatin and gum arabic polymers as wall materials.
According to physicochemical analysis, microcapsules prepared with
greater amount of wall material polymers had slightly smaller mean
particle size. Low moisture content of microcapsules had two positive
effect: 1. Prevented agglomeration thus increasing retention of active
agent, 2. Decreased plasticizer action of water resulted in preservation
of microcapsules glass transition temperature. High encapsulation yield
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LWT - Food Science and Technology 90 (2018) 254–264
was obtained for microcapsules produced with higher wall material
concentration. It was shown that moisture equilibrium results of as-
partame microcapsules were low for all water activities indicating their
high stability, storage and application.
Nori et al. (2011) microencapsulated propolis through complex
coacervation of soy protein isolate and pectin. According to achieved
results, increasing of colloid and core concentrations in solutions de-
creased encapsulation efficiency of propolis indicating that lower in-
teractions established between them. However, on the other hand,
formed microcapsules with high polymer concentrations were a little
more stable than others due to having higher Tg. Finally, authors found
that both antioxidant and antimicrobial activities of propolis were not
significantly changed after applying coacervation and freeze drying
processes. Genipin, a β-glycosidase hydrolysis product of geniposide is a
natural water soluble cross-linker agent. Limitations in the application
of non-toxic cross-linking agents like transglutaminase and tannic acid
in food industry are related to the high price and strange taste, re-
spectively. In a research work, mustard (Sinapis alba) seed essential oil
(containing many glucosinolates, the precursors of the isothiocyanates)
was microencapsulated in gelatin-gum arabic complex coacervates
hardened by genipin. Based on the results of the thermogravimetric
analysis (TGA), the optimum crosslinking time for hardening micro-
capsules was 8 h contributing to higher structural stability. Using ge-
latin as a substrate, genipin showed the highest activity at pH 10.0 led
to hardening of microcapsules with long chains of cross-link bridges. By
increasing the concentration of genipin up to 0.075 g/g gelatin, thermo-
stable microcapsules were produced due to the formation of amide
bonds between genipin ester group and gelatin amino group (Peng
et al., 2014).
Lycopene was microencapsulated within gelatin/pectin complex
coacervates by Silva, Favaro-Trindade, Rocha, and Thomazini (2012).
Spherical microcapsules with a defined wall and core structure were
formed at pH 3.0 resulting in encapsulation of lycopene. However,
drying of microcapsules by freezing or lyophilization processes had
negative effect on stability of coacervates through breaking electro-
static bonds indicating unsuitability of microcapsules for preserving
lycopene during storage. Butstraen and Salaün (2014) micro-
encapsulated commercially available blend of triglycerides (Miglyol
812 N) in gum arabic/chitosan complex coacervates which were
strengthened by the sodium tripolyphosphate as crosslinking agent.
Formation of complexes through electrostatic interactions depends on
the pH of the two solutions and weight ratio of chitosan/gum arabic
mixtures. The optimum pH for coacervation was 3.6 by considering the
high charge of chitosan at pH 2.8–4.0 and negatively charged of gum
arabic above pH 2.2. High biopolymer ratio (opt = weight ratio of
chitosan to gum arabic of 0.25) increased conductivity of the solution
thus resulting in maximum coacervation through neutralization of io-
nized groups of biopolymers at pH 3.6. It was shown that increasing of
the biopolymer volume ratio (r) reduced the mean size of formed par-
ticles with r = 0.10 for microencapsulation process and emulsion time
of 15 min at 11000 rpm as optimum conditions. The calculated work of
adhesion showed the suitability of formed coacervate particles for en-
trapment of core solution due to stronger interactions between gum
arabic/chitosan coacervate microparticles and continuous phase than
between gum arabic/chitosan coacervate and core solution.
Intensity of electrostatic interactions has important effect on the
formation of soluble or insoluble complexes contributing to the pro-
duction of nanoparticles and microparticles. For the preparation of
nanoparticles much stricter conditions are required than the micro-
particles. Strongly negatively charged polysaccharides are not suitable
for the fabrication of the nanoparticles because of forming the insoluble
complexes or precipitated coacervates. Nanoparticles which are more
functional can be formed by mixing proteins and polysaccharides with
moderated charge density (Lv, Yang, Li, Zhang, & Abbas, 2014). Ac-
cording to Jones, Decker, and McClements (2009), globular proteins
and polysaccharides have the potential to produce nanoparticles by
N. Eghbal, R. Choudhary
applying the controlled release process. Complex coacervation method
was applied for the production of heat resistant gelatin/gum arabic
nanocapsules for entrapment of jasmine essential oil by Lv et al., 2014.
It is known that soluble complexes can lead to the formation of nano-
particles. Nanoparticles were formed at pH 4.8 and gelatin/gum arabic
ratio of 1:1 w/w. The pHc corresponding to the onset of soluble com-
plexes formation was defined as the turbidity value (600 nm) changed
from 0 to 0.001 upon acidification by GDL and was constant for all
mixing ratios. At pHφ1 which downshifted as the mixing ratio de-
creased, the formation of soluble complexes was ended. Particle size
distribution analysis revealed that broad polydispersed shape of com-
plexes turned to relatively narrow distribution and then shifted to wide
distribution again as the pH decreased. Nanoparticles were prepared
from the complexes with narrow size distribution centered at approxi-
mately 100 nm. The nanoparticles containing jasmine essential oil were
formed by applying sufficient amount of surfactant coupled with high
shear dispersion processing resulted in formation of system with more
homogeneous nanoparticles. Hardening of formed nanoparticles with
transglutaminase resulted in a slight size reduction also just structural
changes of gum arabic at applied high temperature (80 °C) influenced
the size transition of cross-linked nanoparticles. The volatile com-
pounds of jasmine essential oil could be retained better within nano-
capsules hardened at pH 7.0 during first hour of heating at 80 °C.
However, after 5 h of heating more than 50% of compounds decreased.
They found that nanocapsule size increased through increasing thermal
treatment duration which led to formation of larger pores on the wall
surface and release of compounds. It has been mentioned that fabri-
cation of nanoparticles has attracted great attention as a result of their
high applicability in food, pharmaceutical and biomaterials.
5. Complex coacervates for controlled release of agents
Developing effective delivery systems of bioactives is one of the
main challenges for food producers as the shelf life and the sensorial
quality of enriched foods should not be affected adversely (Moschakis &
Biliaderis, 2017). Microcapsules containing various active compounds
are incorporated in different food products for their antimicrobial and
antioxidant activities, modifying texture and color or masking flavor.
There are few studies relating to the application of microcapsules in
food such as palm oil microcapsules in bread and yoghurt (prepared by
complex coacervation) (Rutz et al., 2017), linseed oil microencapsula-
tion in bread by spray drying method (Gallardo et al., 2013) and ly-
copene microcapsules in cakes (prepared by spray drying method)
(Rocha, Favaro-Trindade, & Grosso, 2012). It is possible to control the
release of encapsulated substances within complex coacervates by pH
which directly influences the stability of coacervates and has similarity
to the reactions that happen in the gastrointestinal tract of human body.
However, besides pH, different parameters such as particle size, struc-
ture, the loading value, the cross-linking degree, temperature and dis-
persing medium affect the controlling release property of coacervate
microcapsules (Nordby, Kjoniksen, Nyström, & Roots, 2003; Rutz et al.,
2017). Recently, palm oil (containing high amount of bioactive agents
especially carotenoids), has been encapsulated by complex coacerva-
tion method using chitosan/pectin (CP) and chitosan/xanthan gum
(CX) as wall materials and different drying methods by Rutz et al.
(2017). Higher encapsulation yield along with lower carotenoid loses
during encapsulation process were obtained for microparticles dried via
lyophilization. Release behaviors of both types of microcapsules were
different in simulated gastrointestinal fluid (GFS) and food. In the
human body, small amount of encapsulated substances can be released
in gastric conditions but the release profile is gradual and complete
under intestinal conditions. In GFS fluid the release rate of active agent
from CP microparticles was higher than CX ones but when applied in
yogurt CX particles had higher release rate and the degradation of re-
leased compounds did not happen in food matrix unlike GFS fluid.
Authors demonstrated that interaction of food matrices with bioactive
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LWT - Food Science and Technology 90 (2018) 254–264
agents can protect them and processing has different effects on each
type of microcapsules.
The effect of different factors including temperature, core/wall
ratio, crosslinking agent on the release of various compounds en-
capsulated in gelatin/gum arabic complex coacervates have been stu-
died (Dong et al., 2011; Peng et al., 2014; Prata, Zanin, Re, & Grosso,
2008; Qv, Zeng, & Jiang, 2011; Rocha-Selmi et al., 2013; Yeo, Bellas,
Firestone, Langer, & Kohane, 2005; Zhang, Zhang, Hu, Bao, & Huang,
2012). Spectroscopic method was used to study the controlled release of
bake flavor oil from gelatin and gum arabic complex coacervates pro-
duced without adding chemical crosslinking agents. Raising tempera-
ture up to 100 °C led to significant release of oil from microcapsules.
But more heat resistant large multivesicular microcapsules having
many small oil cores were produced at higher homogenization rates
(9000 rpm). While low homogenization rates (3000 rpm) resulted in
formation of less heat resistant univesicular microcapsules containing a
single large oil core. On the other hand, at lower concentrations of
polyions, microcapsules with high resistance to release conditions were
produced due to hardly breaking down of formed big agglomerates of
oil droplets. Evaluation of microcapsule stability revealed that integrity
of uncross-linked capsules did not undergo any changes during storage
at 4 or -20 °C for 4 weeks (Yeo et al., 2005).
Increasing concentration of glutaraldehyde as a cross-linker agent
enhanced the cross-linking degree of polymerization resulted in de-
creasing of the oil release rate along with permeability coefficient
(Bachtsi & Kiparissides, 1996). In this relation, changing the con-
centration of transglutaminase (TG, a cross-linker enzyme) and factors
affecting crosslinking process such as hardening time, pH and time have
been suggested to be effective on the release rate of oil from micro-
capsules. Microcapsules containing microalgal oil were synthesized by
complex coacervation of gelatin/gum arabic and the release rate of the
oil was studied as a function of TG crosslinking parameters. Sodium
dodecyl sulfate (SDS) was used as a release medium. The lowest release
rate of oil was achieved for the microcapsules produced under optimum
conditions: hardening time 6 h, temperature 15 °C, pH 6.0 and TG
concentration 15 U/g gelatin. TG showed the highest enzymatic activity
at pH 6.0 contributed to the most hardened microcapsules than those
produced at pH 5.0 and 7.0. Some sustained release of oil was obtained
after the first hour but it displayed a higher rate at the beginning
(Zhang et al., 2012). Dong et al. (2011) investigated the effect of the
core/wall ratio on peppermint oil release from transglutaminase har-
dened gelatin/gum arabic coacervate microcapsules within different
dispersing medium. Larger coacervate microcapsules were formed at
high core/wall ratio resulting in decreased microcapsules membrane
thickness which led to higher release rate in the initial phase. On the
other hand, increased diffusing distance of oil in big particles lowered
its release rate in the later phase of release. It was found that dispersing
medium played an important role on release behavior of oil from mi-
crocapsules. Peppermint oil was slowly released from wet coacervate
microcapsules in hot water through concentration difference between
inside and outside of microcapsules and followed first order release
kinetics model. But oil was released quickly from dried microcapsules
in the oven by vapor pressure difference between both sides of micro-
capsules and followed zero order release kinetics model. Also, storage
stability of coacervate microcapsules were excellent because the release
of core material from cocervates was very slow in cold water with no
significant effect of different core/wall ratios on it.
Peng et al. (2014) studied the effect of relative humidity and tem-
perature on the release rate of mustard essential oil from gelatin/gum
arabic coacervate microcapsules. Remarkable controlled release prop-
erty of round shaped microcapsules could be attributed to multi-
nucleated structure of emulsion droplets with defined walls. In the low
relative humidity environment, microcapsules showed significant re-
duction in oil release however at high humidity rate, they could also
retain high amount of core material compared with non-micro-
encapsulated one. According to the obtained results, formed
N. Eghbal, R. Choudhary
microcapsules with improved stability had the potential of preserving
oil at different temperatures. But it is obvious that high temperature
close to glass transition temperature of microcapsules has deleterious
effect on their structure and stability. Results regarding the effect of
humidity and temperature on microcapsule stability are in consistent
with those obtained by Qv et al. (2011) who microencapsulated lutein
in gelatin/gum arabic complex coacervates. In another work, the effect
of different parameters such as type of crosslinking agent, the release
media and the wet or dry state of microparticles on release of vetiver
essential oil mixed with khusimyl dansylate (KD) as a fluorescent
compound from gelatin/gum arabic micropartcles were studied by
Prata et al. (2008). It was shown that microparticles crosslinked by
glutaraldehyde had smaller sizes after hydration consequently were
more resistant to swelling thus releasing oil slowly compared with
transglutaminase cross-linked microparticles. In fact, polymeric nature
of glutaraldehyde makes it possible to form crosslinking of various
lengths between residues even located far apart. The release properties
of uncross-linked, chemical and enzymatic cross-linked microparticles
containing fluorescent oil also wet and freeze-dried ones were mea-
sured in water, aqueous solution with surfactant and anhydrous
ethanol. Overall, wet microparticles were larger than dried ones be-
cause of hydration contributing to fast release of KD. According to
achieved results, the release of oil was slow with a small initial burst in
aqueous media than alcoholic media.
The release profile of aspartame from gelatin-gum arabic micro-
capsules (2.5% wall material concentration) included two phases with
rapid release in the first phase and slow release in the second phase. The
release rate was high for microcapsules containing high amount of core
material because of having greater mean diameter resulting in in-
creased surface contact with water thus increasing the release rate.
Elevating of temperature had no effect on the release rate of aspartame
which is important to use heat resistant microcapsules for products
needing high temperature processes like chewing gum (Rocha-Selmi
et al., 2013).
6. Conclusion and future perspectives
Important parameters influencing coacervate formation, rheological
properties, microencapsulation of different compounds and controlled
release of agents from coacervates in food related systems have been
reviewed. Defining optimum conditions for obtaining the highest yield
of complex coacervates is crucial in the complex coacervation process.
According to the hypothesis suggested by many researchers, the vis-
coelastic property of coacervates directly depends on the strength of
electrostatic interactions between polymers. But recently, it has been
reported that the highest viscoelastic modulus could be obtained at pH
where the strongest electrostatic interaction did not occur between
charged polymers. More investigations are still needed to better un-
derstand the parameters which affect the rheological property of
complex coacervates.
The mechanical and thermal properties of the microcapsules formed
via complex coacervation can be improved through applying mild
heating, desolvation and cross-linking agents. One of the best methods
for encapsulation of heat-labile agents is complex coacervation process.
Limitations regarding encapsulation of hydrophilic compounds can be
overcame by adding double emulsion step at the beginning. Also, oxi-
dative stability of microcapsules containing lipid extracts could be en-
hanced by changing cross-linking parameters and utilization of dif-
ferent polymers like chitosan (having antioxidant properties).
The application of complex coacervates prepared from biopolymers
in formulated food products has attracted great attention. Capsules
containing active substances can be incorporated in food products with
various purposes including modifying texture and color, showing an-
timicrobial and antioxidant activities. But there are only few studies on
the application of encapsulated agents in food and the release of agents
from coacervates within food matrix. Thus there is a need for greater
263
LWT - Food Science and Technology 90 (2018) 254–264
degree of research on the process and application of complex coa-
cervation in the food industry.
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