International Journal of Biological Macromolecules 121 (2019) 1276–1286

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Review

Complex coacervation: Principles, mechanisms and applications

in microencapsulation
Yakindra Prasad Timilsena a, Taiwo O. Akanbi b, Nauman Khalid b,c, Benu Adhikari d, Colin J. Barrow b,⁎
a
NSW Department of Primary Industries, Yanco Agricultural Institute, Yanco, NSW 2703, Australia
b
Centre for Chemistry and Biotechnology, Deakin University, Geelong, VIC 3217, Australia
c
School of Food and Agricultural Sciences, University of Management and Technology, Lahore, Pakistan
d
School of Science, RMIT University, Melbourne, VIC 3083, Australia

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

Article history: Complex coacervation is a highly promising microencapsulation technique that is extensively employed in phar-
Received 13 September 2018 maceutical, food, agriculture and textile industries. The process involves the interaction of oppositely charged
Received in revised form 19 October 2018 polyelectrolytes in aqueous form. High payload and high encapsulation efficiency (up to 99%), relatively lower
Accepted 19 October 2018
cost of processing, ability to use food-grade shell materials and synthesis at ambient temperature makes coacer-
Available online 22 October 2018
vation an appropriate choice in food and agrochemical industries. Various works have been documented using
Keywords:
different polymer systems and core-shell combinations. This review paper intends to summarize some of the re-
Complex coacervation cent advances in complex coacervation for use in the food and agriculture areas. Current status and future trends
Emulsion of plant proteins utilization for complex coacervation have been reviewed. It is expected that this review will be a
Encapsulation efficiency useful resource for material scientists, food technologists and food engineers.
© 2018 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1276
2. Complex coacervation process and mechanism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1278
3. Theories of complex coacervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1278
4. Factors influencing the coacervation process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1279
5. Methods to optimize and control the complex coacervation process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1281
6. Application of plant proteins and polysaccharides in complex coacervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1282
7. Role of cross linkers in the complexation process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1282
8. Drying of complex coacervates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1283
9. Application of complex coacervation in stabilizing biofunctional oils and oil soluble ingredients . . . . . . . . . . . . . . . . . . . . . . . . 1283
10. Complex coacervation in drug delivery and other areas of biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1283
11. Limitation of complex coacervation microencapsulation process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1284
12. Conclusion and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1284
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1284
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1284

1. Introduction

‘Coacervation’ is a term used in colloidal chemistry to denote the

associative phase separation process induced by modification of the
⁎ Corresponding author. media environment (pH, ionic strength, temperature, solubility) under
E-mail address: colin.barrow@deakin.edu.au (C.J. Barrow). controlled conditions. In this process, the phase which is rich in colloid

https://doi.org/10.1016/j.ijbiomac.2018.10.144
0141-8130/© 2018 Elsevier B.V. All rights reserved.
 Y.P. Timilsena et al. / International Journal of Biological Macromolecules 121 (2019) 1276–1286
+
A
Core dispersed in aq.
solution of anionic Addition of aq. solution
polymer of cationic polymer
Dry Powder
Fig. 1. Schematic representation of complex coacervation process applied in encapsulation of active ingredients.
is known as the coacervate phase, while that containing very little
amounts of colloid is known as the equilibrium phase [1]. Based on
the polymer systems involved in the reaction and the mechanism of
phase separation, there are two types of coacervation processes: these
are simple and complex coacervation. In the simple coacervation pro-
cess, a single polymer is involved and coacervates are formed due to a
dehydration or “water deficit” mechanism caused by the addition of a
salt or desolvation liquid [also called coacervation agent (CA) or induc-
ing agent (IA)] into the reaction medium. A good example of simple co-
acervation process is the phase separation of gelatin when sodium
sulfate (Na2SO4) or ethanol is added into its solution. On the other
hand, in complex coacervation processes, ionic interactions between
two or more oppositely charged polymers, usually proteins and polysac-
charides, leads to coacervate formation and phase separation [2–4]. The
major driving factor for the complex coacervation is electrostatic inter-
action among the charged macromolecules present in the reaction me-
dium. However, van der Waals intermolecular force, hydrophobic
interaction in dipolar proteins and nucleic acid conformation changes
due to intermolecular hydrogen bonding also play important roles in
the complex coacervation mechanism [5–7].
The term coacervation was derived from the Latin word ‘acervus’
(meaning ‘heap’) preceded by a suffix ‘co’ indicating associative phe-
nomenon between two reactants. The term coacervate was first used
in 1623 [1] and coacervation process was first described in 1911 by
F.W.Z. Tiebackx [8,9] and more systematically studied by Bungenberg
de Jong and Kruyt during the 1920s and 1940s using gelatin and gum
Arabic as model biopolymers [10]. The industrial application was first
reported by Barrette Green for the synthesis of carbonless duplicating
paper in 1950s [3,5,10,11].
At present, coacervation is one of the most effective methods of
micro/nano encapsulation used in the food and pharmaceutical indus-
tries [7,11,12]. During the encapsulation process by coacervation
method, the coacervated polymer is deposited around the active ingre-
dient (core) leading to settling down of the encapsulated core [13].
Complex coacervation in food ingredient encapsulation primarily
involves the use of two oppositely charged biopolymers, primarily pro-
teins and polysaccharides, which can form a complex shell surrounding
the core material. Proteins are either of animal origin such as gelatin,
whey proteins, egg albumin and silk fibroin, or of plant origin such as
1277
Change of pH,
temperature or
addition of salt
Interaction
between polyions Hardening/Cross
linking
Drying
Coacervates with
soy protein, pea protein, wheat protein, lentil protein and chia protein.
Similarly polysaccharides include alginate, chitosan, gum Arabic, pectin,
agar, carragenans and carboxymethyl cellulose (CMC). Although gelatin
and gum Arabic are known as the historic biopolymers that were used
for the first time [14] varieties of new pairs of biopolymers have been
utilised for complex coacervation for encapsulating various hydropho-
bic and hydrophilic food and agriculture nutrients and chemicals and
over the last two decades has complex coacervation emerged as a prom-
ising and efficient method of encapsulation of food ingredients such as
omega-3 lipids and sensitive food ingredients [8,15]. Complex coacerva-
tion has been applied to food ingredients [11,12,16–19], pharmaceuti-
cals [12], agriculture [20], textiles [21,22] and electronics [23]. The
application of coacervation processes in agro-production and process-
ing has been mainly limited to the encapsulation of pesticides, phero-
mones and plant extract and oils as insect repellent [24–27].
Coacervation has been increasingly used in the microencapsulation
of industrial ingredients [28–30], although it has also been applied to
the purification of macromolecules such as proteins by selective separa-
tion [31–33] and for the development of innovative and functional ma-
terials by the biopolymer interaction. These novel materials have been
used as fat substitutes, meat analogue and for the synthesis of biode-
gradable films, coatings and packaging materials [33,34]. Recently
Tosgas [35] described Ca2+ ions induced coacervation as a fast method
of detection of the hardness of water.
Complex coacervation is known for its simplicity, low cost, scalabil-
ity and reproducibility in encapsulation of food ingredients that yields
high encapsulation efficiency even at very high payload (up to 99%)
[10,22,36,37]. It has been reported that the characteristics of shell mate-
rials used in the encapsulation process significantly affects the core sta-
bility, process efficiency and degree of protection of active components
[7]. As mentioned before, there are various biopolymers that can be
used in complex coacervation, gelatin in combination with other poly-
saccharides has remained the most widely employed protein source
due to its excellent structural and functional properties. Gelatin is pro-
duced commercially by partial hydrolysis of collagen derived from the
skin, white connective tissues and bones of animals. An acid pretreat-
ment gives type A gelatin while alkali pretreatment produces type B gel-
atin [38,39]. Being produced from the waste material from the meat
processing industry, it is relatively low cost compared with other
1278 Y.P. Timilsena et al. / International Journal of Biological Macromolecules 121 (2019) 1276–1286

biopolymer

Clear
Soluon /
Mixture of
biopolymer Turbid
soluon

Allow to stand
Introducon of without
coacervaon disturbance
agent

Fig. 2. Phase separation in complex coacervation (A: Mixture of biopolymers clear solution, B: Turbid mixture, C: Phase separation showing clearly the coacervate phase and the continuous
phase).

proteins. It is highly soluble in water at elevated temperature (N40 °C)
and forms a clear solution; however, at temperature lower than ambi-
ent, it forms a gel-like structure. Despite the aforementioned advan-
tages, there are certain disadvantages of gelatin. One of them is its
animal origin and its possible association with bovine spongiform en-
cephalopathy (“mad cow disease”), in the case of beef gelatin. Secondly,
the most common form of gelatin, pork gelatin, is not acceptable by cer-
tain group of consumers based on their religious and dietary prefer-
ences. Jenkins et al. [42] and Li et al. [43,44] have reported that plant
proteins are less allergenic than their animal derived counterparts.
Therefore, the search for a plant protein with excellent structural and
functional attributes that can replace gelatin has gained increased inter-
est in the last three decades [40,41]. Section 6 provides a detailed over-
view of proteins and polysaccharides that have been investigated for
this purpose. The major focus of this review is to provide in-depth infor-
mation of complex coacervation and its application in the food and ag-
riculture sectors. Recent advances in complex coacervation technology
used to encapsulate food and bioactive materials are reviewed in details.
2. Complex coacervation process and mechanism
The complex coacervation process is a three-phase system involving
the solvent, the active material and the coating material. In general, this
process involves four steps: (a) preparation of an aqueous solution of
two or more polymers. The solution is generally prepared above the gel-
ling temperature and above the isoelectric point of the protein;
(b) mixing hydrophobic phase to the aqueous solution of one polymer,
usually protein solution, and homogenising the resultant mixture so
that a stable emulsion is produced; (c) change of pH and temperature
to a certain required level to induce coacervation and phase separation;
and (d) hardening of the polymer matrices using elevated temperature,
desolvation agent, or cross-linker [15] as shown in Fig. 1.
The coacervation is initiated by either lowering the temperature or
by altering pH or by dilution or addition of a salt (known as a coacerva-
tion agent). It begins with formation of very fine coacervate particles
giving rise to turbidity in the reacting solution, which otherwise
remains clear or transparent. This stage of coacervation is generally re-
ferred to as micro-coacervation. If the coacervation process is continued,
the microparticles coalesce together so that larger particles are formed.
These larger particles possess higher density so that they tend to settle
at the bottom of the container. After some time, a distinctly visible
two phase system is formed (Fig. 2). This stage is called macro-
coacervation.
Although microcapsule morphology and size are affected by the pro-
cessing conditions, they are generally either mononucleated or multi-
nucleated (Fig. 3). Microcapsules possess a core-shell architecture in
which a small core is completely surrounded by a uniform coating of
the polymer matrix [45]. Lemetter et al. [8] studied the effect of scale
up conditions on morphology and size of microcapsule during sun-
flower oil encapsulation process. They suggested an additional mor-
phology of coacervates called grape bunch type coacervates formed by
the aggregation of mononucleated microcapsules depending upon stir-
ring and gelation conditions. However, the effect of the drying condi-
tions on the morphology of microcapsules was not included in the
study.
3. Theories of complex coacervation
Various theories have been postulated for the coacervation phenom-
enon. Voorn-Overbeek theory, developed on the basis of experimental
Shell
Cores
Core
Fig. 3. Structure of microcapsules from complex coacervation: (A) - mononucleated
capsules, (B) - polynucleated capsule.
 Y.P. Timilsena et al. / International Journal of Biological Macromolecules 121 (2019) 1276–1286 1279

forces among aggregates and coacervates are formed without specific

Fig. 4. Phase diagram of complex coacervation, reprinted by permission [9]. Cp is the total
polymer concentration at a given protein to polysaccharide ratio. The diagram shows close
resemblance to the phase diagram of colloids with attractive interactions. Conductivity
(∼salt concentration) in complex coacervation plays the role of ‘temperature’ in gas–
liquid phase separation, although the phase separation appears to be not dependent on
temperature.
data of Bungenberg de Jong's gelatin/gum Arabic complex coacervation,
is the oldest one [46,47]. Voorn-Overbeek theory states that coacerva-
tion is a spontaneous process driven by electrostatic interaction be-
tween the participating polyelectrolytes. It also describes the effect of
charge densities and molecular weights of polymers, temperature of
ambience, dielectric constant of the solvent and Huggins interaction pa-
rameters on coacervation phenomenon [46]. It describes one step elec-
trostatic interaction where σ3r ≥ 0.53 (σ, charge density and r, polymer
molecular weight) [47]. The phenomenon was better described later by
Veis and Aranyi theory taking Huggins interaction parameter into ac-
count. It also states that coacervation increases significantly on lowering
of temperature [46,48]. The limitation of this theory is that it is applica-
ble to only polymer system of low charge density [46].
Nakajima and Sato further modified Voorn-Overbeek's theory as-
suming that charges are distributed uniformly in both phases during
coacervation-based phase separation. Like Veis-Aranyi's theory, they
also considered Huggins parameter and altered the electrostatic term.
It was developed with the help of oppositely charged pairs of PVA
where σ3r = 0.025 [46–47]. In 1980, Tainaka modified Veis-Aranyi's
theory and postulated that phase separation is driven by attractive
ion pairing [46,47]. Unlike Veis-Aranyi's theory, this is not restricted to
low charge density system. However, both charge density and polymer
molecular weight must lie within a specific range. Among these theo-
ries, Tainaka's theory is considered more practical and applicable to
both low and high charge density systems. However, it fails to provide
the explanation of suppression of coacervation phenomenon at low
ionic strength and complex coacervation process for a multiple polymer
systems [46].
Burgess [48] analysed various complex coacervation systems to re-
solve various contradictory points including the roles of electrostatic
and entropic forces, the significance of Huggins interactions, and type
of charge interaction in the Voorn-Overbeek, Veis-Aranyi, Nakajima-
Sato model and the Tainaka theories. The author studied gelatin/gum
Arabic and gelatin/albumin coacervation and reported the effects of
pH, ionic strength, and polyion concentration in the complex coacerva-
tion process. The author used the microelectrophoretic measurements
to determine optimum pH and ionic strength for complex coacervation.
It was also noted that the effect of ionic strength on complex coacerva-
tion was not the same when total concentration of polyions was altered.
Voorn-Overbeek's theory was inadequate to describe gelatin/gum Ara-
bic coacervation under various conditions whereas the gelatin/albumin
system was found to follow Veis-Aranyi's theory. Thermodynamics and
phase separation mechanisms of the process have been extensively in-
vestigated previously [33,49]. Fig. 4 shows the phase diagram of
model protein-polysaccharide combinations such as gelatin/gum Arabic
and whey protein/gum Arabic systems [9,50,51]. It shows that complex
coacervation and phase separation process depends on the ionic
strength (conductivity) of the solution. Total polymer concentration in
the reaction mixture also plays vital role in enhancing or suppressing
complex coacervation phase separation process. It is clearly shown
that certain concentration of polymer (Cp = 15%, w/w in this case) is
sufficient to induce self-suppression of the complex coacervation
process.
4. Factors influencing the coacervation process
Interactions between participating biopolymers play a significant
role in controlling the structure, texture, and stability of coacervates
and the entrapped material. Therefore, greater understanding of the
influencing factors is critical for better coacervation and its more effi-
cient application [52].
The main driving force for coacervation is the reduction in free elec-
trostatic energy of the reaction system resulting from interaction

Table 1
Effect of pH and biopolymer ratio on complex coacervation.
(Adapted from [127])

Biopolymers pH range Optimum pH Optimum biopolymer ratio References

Gelatin Gum Arabic – 4.0 1:1 [87,88]

Whey protein Gum Arabic 3.0–4.5 4.0 2:1 [40,89]
Soy protein isolate Gum Arabic 2.5–4.5 4.0 1:1 [17]
Chitosan Gum Arabic 2.0–4.0 3.6 4:1 [11]
Gelatin Carboxymethyl cellulose 9.0–11.0 9.0 1:1 [90]
Gelatin Chitosan 4.5–6.5 5.25–5.50 10:1–20:1 [78,91]
Chitosan Carboxymethyl cellulose 3.0–5.0 4.0 [92]
Soy protein isolate Pectin – 4.4 1:1 [93]
Lentil protein isolate Gum Arabic 1.5–8.0 3.5 1:1 [94]
Pea protein isolate Gum Arabic 2.4–4.3 3.6 2:1 [52,95]
Pea protein isolate Chitosan – 6.2 7.5:1 [96]
Pea protein isolate Alginate 1.55–2.98 2.10 4:1 [18]
Alpha gliadins Gum Arabic 2.0–4.0 2.75 3:7 [64]
Pea globulins Gum Arabic 2.0–4.0 3.0 1:1 [64]
Bovine serum albumin Pectin 1.6–4.7 4.7 5:1 [56]
Gelatin Alginate 2.0–5.0 3.5–3.8 3.5:1 [29,63]
β-lactoglobulin Gum arabic 3.5–4.4 3.76 2:1 [97]
Gelatin Pectin 3.8 1:1 [52]
Gum Arabic Chitosan 4.0–5.5 – 5.5:1 [68]
1280 Y.P. Timilsena et al. / International Journal of Biological Macromolecules 121 (2019) 1276–1286

Table 2
Complex coacervation of food and agriculture nutrients.

Core Polymer pairs Polymer mixing ratio pH Cross linker Drying method Encapsulation efficiency (%) Reference

Lemon oil Ge/GA GA 4.2 Glutaraldehyde Solvent washing and drying at room temp 80–90 [98]
Koala flavour Ge/GA GA 4.2 Glutaraldehyde Solvent washing and drying at room temp [98]
Menthol Ge/GA 1:1–1:3 4.5 Glutaraldehyde Spray drying [76]
Lutein Ge/GA 1.25:1 4.2 – Spray drying/freeze drying 85–86 [99]
Eugenol Ge/Alg 4:1 3.5 – – [72]
Jasmine oil Ge/CMC 1:1 4.1 Transglutaminase [100]
Lycopene Ge/GA 4.0 – Dehydration/spray drying/freeze drying [101]

Ge: Gelatin, GA: Gum Arabic, Alg: Sodium alginate, CMC: Carboxymethyl cellulose.

between oppositely charged ions [5]. Temperature, ionic strength and system; however it is less pronounced as compared to that of pH and
pH of the reaction medium, polymers' mixing ratio, their molecular ionic strength [33]. Table 1 summarizes mixing ratio of different bio-
weight, total concentration and charge densities play important roles polymers that result in optimum coacervation. When all the factors
in initiation, continuation and termination of the coacervation process are taken into consideration, it was shown that optimum pH and ionic
[5,6]. Pressure is also an important factor, particularly when supercriti- strength is dependent on mixing ratio of biopolymers, nature of core
cal fluids are used for coacervation [5,53]. Rate of stirring plays an im- material and other experimental conditions [33]. It is evident from
portant role in controlling the size of the coacervates [8]. Furthermore, Table 2 that the same combinations of biopolymers give optimum con-
charges must be sufficiently large to induce interaction, but not large dition at slightly different pH and mixing ratio, depending on the core.
enough to cause precipitation [54]. Highly charged molecules are re- Tables 3 and 4 provide more details on the application of complex coac-
ported to possess extended molecular conformation resulting in ervation in food and agro industries. It can be seen that gelatin is major
unfavourable coacervation [46]. protein ingredient that is widely used in complex coacervation process;
The degree of ionization of the functional groups of proteins (amino however, there are various other proteins that have been investigated
group) and polysaccharides (carboxyl group) is dependent on the pH of for their complex coacervation characteristics with other protein or
the medium in which they exist [33]. Therefore, pH adjustment is essen- polysaccharide pairs. Ru et al. [56] studied the coacervation phenome-
tial in order to initiate the formation of the complex coacervates be- non between BSA and pectin at various biopolymer ratio and pH values
tween majority of protein-polysaccharide pairs [45,55]; however, and found that higher concentrations of protein fraction leads to higher
there are some protein-polysaccharide pairs (e.g. lactoferrin & chia yield of coacervates. However, Xiao et al. [17] demonstrated optimum
seed gum) that can complex instantly when they are mixed together. coacervation was achieved at equal ratio of soy protein isolate/gum Ar-
It is due to the fact that lactoferrin is positively charged protein and abic and changing the ratio significantly altered the coacervation yield.
chia seed gum is negatively charged polysaccharide at normal tempera- It is agreed that shifting of ratio of cationic and anionic polymers from
ture and pH. In general, proteins possess negative charge above their their optimum mixing proportion makes one component deficit and
isoelectric point (pI) but become positively charged as soon as the solu- other component in excess, leaving some molecules of excess polymers
tion pH is dropped below their pIs. This is caused by protonation of unreacted. These unreacted molecules are left over in the equilibrium
amino groups. When an anionic polysaccharide solution is mixed with phase when the coacervates are recovered. Thus, optimization of mixing
a protein solution and pH is reduced below pI of the protein, extent of ratio is also an important consideration from an economic view point.
electrostatic attraction increases due to increased charge difference be- The molecular weights of the participating biopolymers have also
tween the reacting protein and polysaccharide molecules [45]. Firstly, been reported to play an important role in complex coacervation. High
the optimum pH range for highest degree of complex coacervation is molecular weight materials form gels or self-precipitates, whereas low
different for each biopolymer system. Secondly, there is a very narrow molecular weight materials interact with each other by ion pairing
pH range where coacervation is stable between any polymer systems that inhibits coacervation phenomenon [46].
(Table 1) [56]. Some studies reported that temperature used for coacervation also
The quantity of a salt present in the medium affects the ionic plays an important role in the complex coacervation mechanism
strength of the solution, which in turn, influences the complex coacer-
vation process. De Kruif [9] reported that even a small amount of salt
Table 3
weakens the electrostatic binding between polymers. Complete dissoci- Gelatin/Acacia complex coacervation.
ation of newly formed complexes also takes place when salt concentra- (Adapted from [127])
tion is increased [9,56]. However, Weinbreck [57] and Liu et al. [52]
Active Cross Encapsulation Application References
reported contrary results in their studies on the coacervation of whey
ingredient linker efficiency (%)
protein and carrageenan [57]. The authors demonstrated that complex-
Turmeric oleoresin – 49–73 Food [87]
ation between whey protein and carrageenan was enhanced when the
ingredients
concentration of NaCl was small (b45 mM), whereas the complexation Peppermint oil FA or TG 90 Food [102,103]
process was suppressed at higher NaCl concentration (N1 M). It is be- ingredients
lieved that such behaviour is due to the high charge density of carra- Allyl isocyanate TA 84 Oral delivery [81]
geenan [57]. Similarly, complexation between pea protein isolate and Ascorbic acid – 97.3–99.6 Food [104]
Limonene GA 70–86 – [76]
gum Arabic [52] also exhibited no effect at low salt concentration Garlic oil FA 63–99 Food [45]
(≤7.5 mM); however, higher concentration of salt interfered with the Flaxseed oil – – Food [66]
complexation by substantial quantity of pea protein isolate aggregation. Vitamin A FA 64–83 Food [85]
It is also noteworthy that effect of calcium salts (e.g. CaCl2) does not Aspartame – – Food [19]
Dodecanol FA/GA 16–96 Agriculture [105]
have the same effect as that of sodium salts (e.g. NaCl). When CaCl2
α-Terthienyl GA/TA 61 Agriculture [106]
was added to the mixtures, complexes of whey proteins with carra- Microalgal oil TG – – [107]
geenan were formed far above the protein's isoelectric point (up to Vetiver essential GA/TG – – [108]
pH 8) for which calcium bridging was assumed to be responsible [57]. oil
Another important factor that influences the coacervation process is Abbreviations: FA: formaldehyde, TG: transglutaminase, GA: glutaraldehyde, TA: tannic
the mixing ratio and the final concentration of biopolymers in the acid.
 Y.P. Timilsena et al. / International Journal of Biological Macromolecules 121 (2019) 1276–1286 1281

Table 4 5. Methods to optimize and control the complex coacervation

Gelatin and biopolymers in complex coacervation. process
Polymer Core Encapsulation Drying References
combinations efficiency (%) method As described in Section 4, the complex coacervation process be-
Gelatin Chitosan Zanthoxylum 41–81 NR [78] tween various biopolymers is affected by material properties as well
limonella oil as the process parameters. The material properties include chemical
Gelatin Pectin Lycopene 90–93 Freeze [109] composition and functional properties of polymers, their molecular
drying
weights, charge densities, molar concentrations in solution and weight
Gelatin Guar gum Clove oil 50 Vacuum [110]
drying ratio of each polymer. Process parameters include method of emulsion
Gelatin Alginate Ascorbic acid 98 Freeze [104,111] preparation such as homogenization rate, pressure and time, pH, ionic
Eugenol 6–16 drying [72] strength and temperature of the medium, and cross-linking and drying
– methods employed. Furthermore, these influencing factors are interde-
Gelatin CMC Neem oil 72–97 Freeze [112]
drying
pendent on each other. Therefore, an optimized condition for one set of
Gelatin Carbopol Sulphadiazine 60–85 Vacuum [113] polymers cannot be exactly replicated for another set of polymers. That
drying is why optimization of the coacervation parameters is important for
Gelatin Carrageenan Neem seed oil 76–96 Freeze [77] each specific system, prior to its application in microencapsulation of
drying
bioactive ingredients.
Abbreviations: NR: not reported, CMC: carboxymethyl cellulose. Several methods have been explored to characterise coacervation
process and optimize coacervation conditions for various types of poly-
[46,56]. Low temperature enhances the coacervation process because of mers (Table 5). Tsung and Burgess [54] employed coacervate yield and
the increase in solvent-solvent, solvent-solute and solute-solute microelectrophoretic mobility of individual biopolymers to study the
interactions. However, other studies showed insignificant temperature coacervation between heparin and gelatin and find the optimum coac-
effect. de Kruif [9] reported that entropy gain is the main driver in com- ervation conditions. The authors reported that maximum coacervate
plex coacervation and the process is independent of the temperature. yield was obtained at pH 2.6 with ionic strength of 10 mM and hepa-
High concentration of biopolymers also exhibits a negative effect on rin/gelatin mixing ratio of 1:2. Koh and Tucker [62] studied complex co-
coacervation. Burgess [46] indicated that high concentration does not acervation of sodium carboxy-methylcellulose and gelatin system using
allow the free movement of the molecules and restrict them in close viscometric analysis. The authors reported that optimum coacervation
proximity, reducing the energy gain during coacervation. This restricted was found at pH 3.5 with a polymer weight ratio of 3:7. Shinde and
movement suppresses interactions between macromolecules. Nagarsenker [63] also characterized coacervation conditions based on
Previous studies [17,45,58] reported that processing parameters and viscometric and turbidometric analysis and measuring the dry yield of
techno-functional aspects of the system also affect coacervation condi- coacervates in gelatin-alginate system. Optimum coacervation was
tions and coacervate phase properties (such as rheology and yield), achieved at pH 3.5 with gelatin/alginate ratio of 4:1.
and encapsulation efficiency of specific compounds [8]. The effect of In recent years, measurement of zeta potential of individual poly-
processing parameters (shear, temperature and residence time) on mers and the reaction mixture has been used as a tool to elucidate coac-
the final product characteristics have not been well described in the lit- ervation process and optimize the conditions in several biopolymer
erature [8]. Interfacial tension, viscosities and densities of the reaction systems. Since zeta potential varied with pH of the reaction medium,
mixture also influence complex coacervation process [11]; however, optimum coacervation occurs when the charge difference between par-
these aspects are also poorly reported in literature. ticipating polymers is at maximum value [64]. Liu et al. [52] reported a
Other factors influencing complex coacervation-based microencap- correlation between turbidity and zeta potential in their study on com-
sulation of bioactive food ingredients are reported in some previous plex coacervation of pea protein isolate (PPI) and gum Arabic (GA). The
studies [10,59]. Gosh [10] pointed out that agglomeration of microcap- authors reported that a PPI/GA ratio of 2:1 produced coacervates with
sules severely hampers the success of the complex coacervation pro- net neutral surface charge (zeta potential ~0 mV) at pH of 3.57 and tur-
cess; however, careful control over pH, temperature and agitation bidity (absorbance) reached a maximum at that point. Wang et al. [65]
conditions can easily resolve this problem. Similarly, Xing et al. [59]
studied the effect of surfactants on coacervation-based microencapsula-
tion of capsaicin. The authors reported that surfactant rigidifies micro- Table 5
Methods used to characterise complex coacervates.
capsule structure and thus increases the yield and oxidative stability
of the microencapsulated product; however, there was no mention Biopolymer pairs Characterisation methods used Reference
about the effect of surfactant on the coacervation phenomenon. In an- Heparin and gelatin Coacervate yield and [54]
other study, Mayya et al. [60] noted that addition of oppositely charged microelectrophoretic mobility
surfactant significantly increased the yield of coacervates. It was also Sodium Viscometric analysis [62]
carboxymethylcellulose-gelatin
suggested that surfactant concentration was crucial during microencap-
Gelatin-alginate Viscometric and turbidometric [63]
sulation of paraffin oil using a two-layer encapsulation process. The sur- analysis and coacervate yield
factant firstly interacts with oppositely charged polymers, forming a Pea protein isolate-gum Arabic Turbidity and zeta potential [52]
primary layer and contributes in reducing surface tension at oil/water Gelatin and sodium Turbidity, zeta potential and [65]
interface. A second layer of polyelectrolyte–polyelectrolyte complexa- hexametaphosphate coacervate yield
Chia seed protein isolate-chia Turbidity, zeta potential and [116]
tion is formed thereafter between oppositely charged components. seed gum coacervate yield
Rabišková and Valášková [61] also studied the influence of surfactants Flax protein isolate-flaxseed gum Turbidity, zeta potential and [117]
on the encapsulation efficiency of complex coacervation-based micro- coacervate yield
encapsulation processes using three different oils as core materials. It Whey protein isolate-gum Arabic Turbidity, zeta potential and [118]
coacervate yield
was reported that addition of surfactants during emulsion preparation
Whey protein-gum Arabic Optical microscopy [40]
affect the encapsulation efficiency and the effect of surfactants Sodium Chemical analysis of coacervate [67]
depended on the hydrophilic lipophilic balance (HLB) values of the sur- carboxymethylcellulose-gelatin and equilibrium phase
factants. Surfactants with HLB values between 1.8 and 6.7 resulted in Gum Arabic and chitosan Zeta potential and viscoelastic [68]
maximum uptake of the emulsion into the complex coacervate properties
Pea proteins-gum Arabic Interfacial properties [69]
microcapsules.
1282 Y.P. Timilsena et al. / International Journal of Biological Macromolecules 121 (2019) 1276–1286
Table 6
Animal proteins for complex coacervation.
Polymer Core Encapsulation Drying References
combinations efficiency (%) method
WP GA Dodecyl acetate 41–87 Vacuum [114]
drying
WP GA Sunflower oil, 80–90 Directly used [40]
lemon oil, orange oil in Gouda
flavour cheese
CH CS Lavender oil 37–60 Freeze drying [115]
Abbreviations: WP: whey protein, BSA: bovine serum albumin, GA: gum Arabic, CMC: car-
boxymethylcellulose, EPS: extracellular polysaccharide, EA: egg albumin, BLG: β-lacto-
globulin, CH: collagen hydrolysate, CS: chitosan.
also employed zeta potential and turbidity measurement to illustrate
coacervation conditions of gelatin and sodium hexametaphosphate
and used this system for the microencapsulation of tuna oil. Recently
Timilsena et al. [116], Kaushik et al. [117] and Eratte et al. [118]
employed the same method to characterise complex coacervation be-
tween chia seed protein isolate (CPI)-chia seed gum (CSG), flax protein
isolate (FPI)-flaxseed gum (FG) and whey protein isolate-gum Arabic.
Timilsena et al. [116] reported that CPI and CSG yield the highest quan-
tity of complex coacervates at a pH of 2.7 and a mixing ratio of 6:1. Sim-
ilarly, Kaushik et al. [117] found optimum conditions for complex
coacervation between FPI and FG at pH 3.1 and mixing ratio of 3:1.
Several researchers have used optical microscopy [40,64,66] for
assessing the optimum condition of complex coacervation between
numbers of biopolymers. Weinbreck et al. [40] employed simple optical
microscopy techniques to observe the complexation and oil encapsula-
tion by whey protein (WP)/gum Arabic (GA) system. It was observed
that above pH 5.0 no oil was encapsulated in the WP/GA complex coac-
ervates. In the pH region from 4.5 to 3.0, a layer of coacervate was seen
around the oil droplets. When the pH was dropped to 4.0, a smooth co-
acervate layer is formed surrounding the oil droplet completely
entrapping it. It was also reported that oil encapsulation by the complex
coacervates was highest at pH 3.5 and almost nil at pH values lower
than 3.0. This all corroborated that complex coacervation between
protein and polysaccharides occur within a specific and narrow pH
window.
Koh and Tucker [67] characterized the complex coacervation phe-
nomenon between sodium carboxymethylcellulose (SCMC) and gelatin
by chemical analyses of the coacervate and the equilibrium fluid phases.
It was found that optimum coacervation between SCMC and gelatin
takes place at mixing ratio of 3:7 and pH values of 3.5. In a study,
Espinosa-Andrews et al. [68] reported interrelationship of zeta potential
and viscoelastic properties during complex coacervation of gum Arabic
and chitosan. The authors found a clear interrelationship between tur-
bidity, size and zeta potential of the coacervate mixture with the coacer-
vate yield. The highest coacervate yield (~88%) was obtained with a
gum Arabic/chitosan ratio of 5.5:1 and pH of 4.5, when the charge differ-
ence between participating polymers was maximal. Viscoelasticity
measurement at different biopolymer ratios was performed to deter-
mine percent strain and inflection point in the storage and loss moduli
and inflection in moduli was considered the breakdown point of the
complex coacervate structure. However, the pH was assumed to be con-
stant irrespective of its shift in moduli as reported in other literature
[52,56].
Interfacial and surface tension properties are useful in determining
the optimum coacervation and phase separation kinetics for various
biopolymers. However, there is not very much published literature in
this area [69–71]. Ducel et al. [69] demonstrated an interrelation of
the interfacial properties of plant proteins-gum Arabic mixing ratio
and coacervation during encapsulation of vaselin oil. The effect of pH
on the ability of the coacervate phase to form a layer around oil droplets
was studied using pendant drop tensiometry. It was reported that lower
pH conditions created an interfacial viscoelastic behaviour indicating
softness of the interfacial film which is advantageous in forming a con-
tinuous layer around the oil droplets to be encapsulated.
6. Application of plant proteins and polysaccharides in complex
coacervation
Although gelatin has been extensively used as a protein source in
complex coacervation process [29,63,72] with a range of carbohydrate
polymers (e.g. gum Arabic and sodium alginate) association of beef gel-
atin with prion disease and the animal origin of gelatin in general has re-
sulted into its reduced popularity. Consequently, other animal proteins
(Table 6) and various plant proteins have been explored as an alterna-
tive to gelatin in various food product developments and as shell mate-
rial in complex coacervation-based microencapsulation technologies
(Table 7).
Studies also suggest that appropriately processed plant proteins, ex-
cept for a few (e.g. gluten and peanut protein), are comparatively less
allergenic in comparison to gelatin and other animal proteins (e.g.
milk proteins and egg proteins) [42,43]. Due to an increased popularity
of plant proteins and their healthier nature, exploration of newer vege-
table proteins has been growing in recent years. Their suitability in dif-
ferent food and non-food applications has been explored and reported.
Advances in plant proteomics has also contributed significantly to the
scientific understanding of the composition and benefits of these high
value protein sources.
Plant proteins are produced commercially in the form of concen-
trates or isolates. These protein forms are composed of different frac-
tions such as albumin (water soluble), globulin (soluble in salt
solution), glutelin (soluble in alkaline aqueous solutions) and prolamin
(soluble in water and ethanol). These different fractions are usually sep-
arated by differential solubility methods. However, in most of the labo-
ratory scale plant protein isolates have been used that were obtained by
alkali solubilisation and isoelectric precipitation method [73].
Among various plant proteins, soy protein is the most well studied
and most commonly employed plant proteins; however, other plant
proteins such as pea protein isolate, lentil protein isolate, canola protein
isolate, sun flower protein isolate and wheat protein isolate are also be-
coming popular in recent years. It is also reported that fractions of soy
proteins (e.g. glycinin and conglycinin) and pea proteins (e.g. legumin
and vicilin) have also been investigated for their application in microen-
capsulation of different food ingredients [74]. Recently, Timilsena et al.
[116] and Kaushik et al. [117] employed chia seed protein and flaxseed
protein isolates to study their complexation behaviour with chia seed
gum and flax gum, respectively.
7. Role of cross linkers in the complexation process
Cross linkers are generally used to obtain stable microcapsules with
improved thermo-mechanical properties. In many cases, application of
mild heat is also used to stabilize the complex coacervate microcapsules
[75]. Traditionally, formaldehyde, glutaraldehyde, glyoxal, diisocyanate,
epichlorohydrin and polyamines have been used as cross-linking
Table 7
Plant proteins and polysaccharides in complex coacervation.
(Adapted from [127])
Polymer Core Opt Polymer EE (%) Drying References
combinations pH ratio method
SPI/GA Sweet orange 4.0 1:1 65–78 Spray [17]
oil drying
SPI/pectin Casein 4.4 1:1 79–92 Freeze [93]
hydrolysate drying
SPI/pectin Propolis 4.0 – 66–72 Freeze [30]
extract drying
Abbreviations; SPI: soy protein isolate, GA: gum Arabic, PPI: pea protein isolate, GG: guar
gum.
 Y.P. Timilsena et al. / International Journal of Biological Macromolecules 121 (2019) 1276–1286
agents. In chemically-induced cross-linking process, a non-soluble net-
work is formed via the reaction of aldehyde residues from the cross-
linking agent and amino groups from the protein. This newly formed
network strengthens the capsule wall, thereby facilitating drying pro-
cess and increasing storage stability of the capsules [76]. Although
these chemical cross-linkers are highly efficient in stabilizing protein-
based or complex coacervates-based microcapsules, their application
in food is limited due to their reported toxicity. This has necessitated
the exploration of novel and non-toxic cross-linkers to be used in food
industries. As a consequence, genipin [77,78], sodium tripolyphosphate
(NaTPP) [79,80] and tannic acid [81] have been used in cross-linking.
Another notable cross-linkers, transglutaminase, an enzymatic cross
linker, has also been used in various food formulations and microencap-
sulation processes. Dong et al. [82] optimized the cross-linking parame-
ters while producing transglutaminase (TG)-hardened spherical
multinuclear microcapsules (SMMs) by complex coacervation with
gelatin–gum Arabic as shell material and peppermint oil as the core.
The authors found that hardening time of 6 h at 15 °C and at pH 6.0
with a TG concentration of 15 U/g of gelatin produced the best effect.
It was also reported that TG exhibits similar microcapsule hardening ef-
fectiveness to that of formaldehyde.
Leclercq et al. [76] reported that temperature of reaction medium
also plays an important role in the efficacy of cross linkers. Cross linking
of the gelatin/gum Arabic microcapsules with glutaraldehyde promotes
reaction between an aldehyde group and amino group better at alkaline
conditions (pH ~9) than at acidic pH values. This was supported by en-
hanced oxidative stability of limonene oil after microencapsulation.
8. Drying of complex coacervates
Drying is essential to obtain the coacervated microcapsules in pow-
der form for their further application in food formulations. However,
some food applications do not require dry capsules and microcapsules
containing functional ingredients, as the core material can directly be
incorporated in food products [40]. Previous studies on complex coacer-
vation primarily focused on optimization of capsule formation process;
however, the effect of drying conditions on physicochemical nature of
capsules and the encapsulated core are not well studied. Literature sug-
gests that coacervates are recovered from the reaction mixture by sim-
ple filtration, dehydrating in alcohol or silica and by oven drying,
vacuum drying, spray-drying or freeze-drying. Table 2 shows that freeze
drying and spray drying are two most commonly employed drying
techniques; however, both methods have some advantages and disad-
vantages. In general, spray drying yields nearly spherical particles of
5–50 μm size, whereas freeze drying produces a dried lump that must
be ground to a powder form by mechanical disintegration. During this
grinding process, some capsules break down and a portion of the core
may get leached out. This core material that comes to the surface of
the capsule and is exposed to the external environment is more prone
to degradation (oxidation). In principle, freeze drying uses lower tem-
perature to dry the microcapsules. Therefore, thermally sensitive actives
are usually dried using freeze drying method in order to minimise deg-
radation by heat [83]. Timilsena et al. [119] studied the comparative ef-
fectiveness of drying on capsule morphology and core stability and
reported that chia seed oil microcapsules obtained by spray drying pos-
sessed higher oxidative stability than those obtained from freeze drying.
Palmieri et al. [84] compared the efficiency of three drying methods
(isopropanol dehydration, spray drying and freeze drying) on microen-
capsulation of a drug. They also recommended spray-drying to yield
capsules with better morphology and load retention. However, the ef-
fect of drying conditions on core stability was not investigated in this
study. A similar study carried out by Laclercq et al. [76] showed that
freeze-drying was the superior method to produce free-flowing, more
intact, lower moisture (~3%) particles. In another study, Junyaprasert
et al. [85] prepared microcapsules of vitamin A palmitate using
gelatin-acacia complex coacervation and recovered dry microcapsules
1283
using air drying (hot air at 40 °C) and freeze-drying methods. The au-
thors recommended that freeze-drying yielded microcapsules with ex-
cellent properties. Overall, spray drying is the preferable method due to
ease of scale up and low cost, compared with freeze drying, for materials
that are not to temperature sensitive.
9. Application of complex coacervation in stabilizing biofunctional
oils and oil soluble ingredients
Biofunctional oils containing essential long-chain polyunsaturated
fatty acids (LC-PUFAs) are mostly marine-derived. These include fish,
krill and microalgal oils [120–122]. The most widely reported LC-
PUFAs are eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA). These highly oxidatively unstable fatty acids have been reported
to have a broad range of health benefits. They are known to play a role in
nervous system activity, cognitive development, memory-related learn-
ing, neuroplasticity of nerve membranes, synaptogenesis and synaptic
transmission [123]. As a result, research efforts have been focused on
developing methods of stabilizing these fatty acids including microen-
capsulation [124–129]. Microencapsulation is known to be highly effec-
tive in preventing deterioration of oils because it builds protective
barriers around oils thereby preventing the formation of unpleasant
taste, flavour and odour [127,129,130]. Different types of microencapsu-
lation techniques such as spray/freeze drying, coacervation, submerged
co-extrusion, liposome entrapment and melt injection have been re-
ported [129]; however, spray drying technique remains the most
widely reported owing to its low cost, flexibility and ease of scale up
[131]. Interestingly, microencapsulation by complex coacervation has
been reported as the most successful technique because it allows the
development of microcapsules with a multi-core structure [131]. It has
also been found to significantly stabilize fish oil against oxidation than
simple spray drying because oil resides closer to the centre of the parti-
cle and a thick outer shell is present providing better protection against
oxygen penetration in coacervated microcapsules [124,128].
The oxidative stability of tuna oil was significantly increased after
microencapsulation using multicore complex coacervation technique
[128]. This technique was also reported to prevent degradation of
other heat-sensitive bioactive compounds, such as vitamins A, D3, E,
K2, curcumin and coenzyme Q10 microencapsulated with tuna oil
[130]. A recent study found that lipase-produced concentrates of
omega-3 fatty acids from anchovy oil showed significantly high stability
when encapsulated using complex coacervates of gelatin and sodium
hexametaphosphate (SHMP) [126]. Morphological and physicochemi-
cal analysis indicated that the microcapsule from concentrates exhibits
significantly smoother surfaces and improved mechanical strength
compared to the microcapsules fabricated from native anchovy oil
[126].
10. Complex coacervation in drug delivery and other areas of
biotechnology
Complex coacervation is one of the oldest and simplest method for
encapsulating drugs [132]. Drugs such as metronidazole hydrochloride,
diclofenac sodium and indomethacin have been encapsulated using
complex coacervation [133]. Controlled delivery of these drugs was
studied using pectin-gelatin and alginate-gelatin complexes [133]. Pep-
sin/casein microcapsules were prepared by complex coacervation to en-
able prolonged release of acetaminophen, a drug used to relieve pain
and reduce fever [134]. Authors found that the release of acetamino-
phen from the microcapsules was slow and gradual, suggesting that
this technique is appropriate for a sustained release of acetaminophen
throughout the gastrointestinal tract [134].
This technique has also been used in other areas of biomedical and
biotechnological research. For instance, alginate-chitosan complex co-
acervation for kidney cell encapsulation has been reported. This tech-
nique achieved high cell viability and improved mechanical properties
1284 Y.P. Timilsena et al. / International Journal of Biological Macromolecules 121 (2019) 1276–1286
but requires validation through clinical application [135]. Complex co-
acervation has been used to produce microcapsules of lutein with im-
proved stability against light, humidity and temperature [99]. This
technique has also been used for the microencapsulation of flavours
and thermally sensitive compounds [136].
11. Limitation of complex coacervation microencapsulation process
Due to high payload efficiency and ambient temperature process-
ing, complex coacervation is an appropriate choice for micro/
nanoencapsulation of a range of ingredients. The microparticles ob-
tained by complex coacervation are completely surrounded by
shell matrix and thus protects the core from damage from external
stressors such as moisture, light and oxidation. However, one of the
major limiting factors for the commercial application of complex co-
acervation technology is its sensitivity to pH and ionic strength [86].
Complex coacervation takes place within a very narrow pH window,
usually below the isoelectric point of the participating protein, so it is
usually difficult to protect low pH sensitive material during encapsu-
lation. Furthermore, the presence of small amounts of salt can cause
dissociation of complex coacervates, requiring salt free condition
(e.g. deionized water for the preparation of polymer solutions).
12. Conclusion and future perspectives
Although several methods of microencapsulation of biofunctional
compounds have been reported, complex coacervation remains the
only technique with high payload and high encapsulation efficiency
up to 99%. Besides, unlike other techniques, complex coacervation al-
lows the development of microcapsules with multi-core, thus offering
greater protection for biofunctional compounds against degradation
during processing and storage. This technique has been successfully
used for encapsulating a broad range of biofunctional ingredients in-
cluding omega-3 oils, polyphenols, flavours, fat-soluble vitamins and
pigments. Several studies have shown that compounds encapsulated
by complex coacervation technique displayed remarkable oxidative sta-
bility with improved controlled-release behaviour.
Research on complex coacervation has been in practice for approxi-
mately 6 decades, with growing theoretical and experimental knowl-
edge on newer wall materials/core compositions. Growing concerns
with gelatin has led to exploration of plant based material for the pro-
duction of engineered foods with functional properties, but the solubil-
ity issues of plant proteins has sometimes resulted in poor product
formation. The effect of processing parameters on the core stability
and its release behaviour and application of natural cross-linker such
as genipin, tannic acid and transglutaminase to replace traditionally
used potentially toxic cross-linkers, such as glutaraldehyde and formal-
dehyde, require further study.
Acknowledgements
This work was partially supported by Australian Research Council's
Linkage Project (LP140100722).
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