01 复合凝聚法微胶囊化富含多不饱和脂肪酸 (Pufas) 的植物油的研究进展
01 复合凝聚法微胶囊化富含多不饱和脂肪酸 (Pufas) 的植物油的研究进展
Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd
a r t i c l e i n f o a b s t r a c t
Article history: Polyunsaturated fatty acids (PUFAs) are essential fatty acids that are abundantly available in marine fish,
Received 28 October 2016 algae and some plant seeds. In recent years, the demand of plant-based PUFAs is increasing rapidly due
Received in revised form to dietary and lifestyle requirements. The toxicity and environmental sustainability concerns associated
9 February 2017
with fish oils are driving the demand for plant-based PUFAs. PUFAs-rich oils rapidly undergo deterio-
Accepted 2 March 2017
Available online 6 March 2017
ration due to oxidation in the course of processing, transportation and storage. They require adequate
protection and are microencapsulated before their application or incorporation in food products. Com-
plex coacervation-based microencapsulation technology is increasingly being used in food and phar-
Keywords:
Polyunsaturated fatty acids (PUFAs)
maceutical industries due to high encapsulation efficiency, high payload and mild processing conditions
Plant oil associated with this technology. This article provides a comprehensive overview of the relevant scientific
Microencapsulation literature on complex coacervation with special emphasis on the production of plant protein-plant
Complex coacervation polysaccharide (gum) complex coacervates. Microencapsulation of PUFAs-rich oils in plant protein-
Digestion gum complex coacervates and resultant oxidative stability of PUFAs is highlighted. Finally, the release
and digestion of encapsulated PUFAs in the human physiological conditions are discussed. Co-
encapsulation of various functional ingredients together with PUFAs derived from plants in plant
protein-gum complex coacervate is identified as important future research direction.
© 2017 Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
2. Fatty acids and PUFAs profile and content in plant oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
3. Complex coacervation process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
4. Microencapsulation of PUFAs-rich oil using complex coacervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
5. Recent advances in complex coacervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
6. Physiological digestion of encapsulated oils and fats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
7. Concluding remarks and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
1. Introduction
http://dx.doi.org/10.1016/j.foodhyd.2017.03.007
0268-005X/© 2017 Elsevier Ltd. All rights reserved.
370 Y.P. Timilsena et al. / Food Hydrocolloids 69 (2017) 369e381
nutrients. PUFAs play vital role in maintaining health and wellbeing compounds and causes rancidity in foods (German, 1999). There-
in humans by minimizing the risk of cardiovascular and neurode- fore, suitable protection and delivery mechanisms have to be
generative disease, arthritis, diabetes and certain types of cancer developed in order to prevent the degradation of PUFAs-rich oils
(Orsavova, Misurcova, Ambrozova, Vicha, & Mlcek, 2015). Since during processing, storage and transportation and to facilitate their
human body is unable to synthesise PUFAs in required quantity, incorporating in processed foods.
they are known as the essential fatty acids (EFAs) and have to be Two main strategies are being pursued by food industries to
supplied to the body through diet or dietary supplements. minimise the deterioration of PUFAs-rich oils during processing,
PUFAs are abundantly present in marine fish, algae and some transportation and storage. They are: 1) incorporation of natural or
plant seeds. PUFAs contained in plant oils are short chain fatty acids synthetic anti-oxidants in oils and fats to minimise oxidation, and
(SCFAs) such as a-linolenic acid (ALA, C18:3 u 3) and linoleic acid 2) microencapsulation of oils and fats to prevent their direct con-
(LA, C18:2 u 6), whereas fish and algal oils contain long chain tact with environmental stressors including oxygen, heat, moisture
PUFAs (LC PUFAs) such as eicosapentaenoic acid (EPA, C20:5 u 3), and light. In many cases, both strategies are applied concurrently to
docosapentaenoic acid (DPA, C22:5 u 3), docosahexaenoic acid achieve synergistic effects. This review will focus only on the sec-
(DHA, C22:6 u 3) and arachidonic acid (AA, C20:4 u 6). It has ond strategy.
been commonly accepted that these LC PUFAs can be synthesised in Microencapsulation is a process during which a core material
human body using SCFAs (ALA and LA) as precursors, provided they (e.g. PUFAs-rich oil) is entrapped inside a thin layer of coating
are present in sufficient quantity. Therefore, ALA and LA are known material (shell). Protein and polysaccharides including gums, either
as true “essential” fatty acids (Mehta, 2006; Rubio-Rodríguez et al., individually or in complexed form, are commonly used as shell
2010). It is also established that the extent or percentage of con- materials for microencapsulation of food ingredients including
version of ALA to EPA and DHA and that of LA to AA is small, for PUFAs-rich oils. The shell matrix creates a physical barrier between
example, only 15e25% of ALA is converted into DHA in human body the core and the external environment so that the core is prevented
(Burdge & Wootton, 2002; Emken, Adlof, & Gulley, 1994). It is also from direct contact of moisture, oxygen, heat, light, acid or alkali.
believed that this slower conversion process does not produce The shell matrix also helps to mask the undesirable odour, taste or
sufficient LC PUFAs to meet the physiological demand in human colour of the core so that sensory attributes and palatability of the
body and thus EPA and DHA should also be supplied in the diet in product are either enhanced or at least not compromised.
order to maintain health and well-being. Due to these reasons, EPA Furthermore, shell can also prevent undesirable interaction be-
and DHA are also considered as EFAs. tween core material and other food components and can enhance
Commercially available long chain PUFAs mostly come from fish their bioavailability (Sanguansri & Augustin, 2011). In recent years,
oils derived from fatty fish such as tuna, sardine, mackerel, herring microencapsulation is used to design controlled release formula-
and trout (Meyer et al., 2003). However, these fish oils possess tions in order to achieve targeted and sustained delivery of lipids,
strong fishy flavour and are not favoured by consumers and cannot increase satiety and help control the body weight (Ubbink &
be incorporated into vegetarian and vegan diets. Furthermore, Krüger, 2006; Wilson & Shah, 2007). Thus, microencapsulation
current global production of fish is unable to meet the demand of enhances the nutritional efficacy of food additives and helps
u-3 PUFAs which is of the order of 1.25 million metric tonnes per broadening the application of unstable food ingredients in a wider
annum (Betancor et al., 2015). Due to the high demand of PUFAs- range of food products (Desai & Park, 2005). Microencapsulation
rich fish oil, marine fish has been harvested excessively, which if has become an important and ubiquitous technology for stabilizing
not managed correctly, would result in the decline and eventually and delivering numerous unstable and lipophilic ingredients in
extinction of these fish species. Furthermore, there is an increasing food and pharmaceutical products.
concern of the presence of methyl mercury and organic pollutants The first successful microencapsulation of flavour oil was carried
such as polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated out in gum acacia matrix through spray drying in 1927 (Fanger,
dibenzofurans (PCDF) and dioxin-like polychlorinated biphenyls 1974). Spray drying is now more frequently and widely used in
(DL-PCB) in marine fish (Smutna, Kruzikova, Marsalek, Kopriva, & industry to encapsulate many unstable food ingredients and active
Svobodova, 2008). Hence, many food manufacturers and re- pharmaceutical agents (Arslan, Erbas, Tontul, & Topuz, 2015).
searchers are interested in developing alternative source of PUFAs Several other methods such as freeze drying (Anwar & Kunz, 2011),
in order to address the aforementioned limitations in the inherent fluidized bed coating (Dewettinck & Huyghebaert, 1998), centrif-
characteristics and availability of fish oils. ugal extrusion (Desai & Park, 2005), inclusion complexation (Song,
One of the important considerations in selecting ideal source of Bai, Xu, He, & Pan, 2009), complex coacervation (Wang, Adhikari, &
PUFAs is the ratio of u-6 to u-3 in the oil. This ratio is very Barrow, 2014; Eratte et al., 2015; Timilsena, Wang, Adhikari, &
important because heavily processed western diets are rich in u-6 Adhikari, 2016; Timilsena, Adhikari, Barrow, & Adhikari, 2016),
PUFAs but deficient in u-3, thus creating a gross imbalance in the ionotropic gelation (Bokkhim, Bansal, Grøndahl, & Bhandari, 2016),
intake of u-6 and u-3 (De Silva, Francis, & Tacon, 2011, pp. 1e20). In liposome entrapment (Mozafari et al., 2008), and electro-spraying
order to address this issue, consumption of oils containing higher (Coghetto et al., 2016) have also been developed in order to
concentration of u-3 is to be increased. As a consequence, demand encapsulate large array of unstable food and pharmaceutical
of foods enriched with u-3 is rapidly increasing. However, incor- components. The choice of microencapsulation methods depends
poration of these u-3 PUFAs in processed foods still remains chal- on the nature of the core and shell material and intended appli-
lenging due to their unstable nature. cation of the final product. The design of an effective encapsulation
The chemical structure of PUFAs [Fig. 1 (AeE)] shows they have system requires a sound understanding of the physicochemical
two or more double bonds in their structure. PUFAs are comprised mechanisms of encapsulation, possible interaction of the active
of bis-allylic methylene groups (eCH]HeCH2eCH]CHe) and all compounds with the shell matrix and the nature of shell matrix
the double bonds are present in the cis-configuration. This type of (Augustin & Hemar, 2009).
chemical structural make up renders them inherently unstable and Complex coacervation is regarded as one of the simplest, low
sensitive to oxidation, isomerisation and polymerisation when they cost, scalable, reproducible and most effective methods of micro-
come in contact with environmental stressors such as oxygen, encapsulation of hydrophobic substances (Barrow & Shahidi,
moisture, heat, light and metallic ions (Rustan & Drevon, 2005). 2007). This method yields considerably high encapsulation effi-
Oxidation of lipids produces undesirable and harmful volatile ciency and low surface oil and provides high oxidative stability of
Y.P. Timilsena et al. / Food Hydrocolloids 69 (2017) 369e381 371
Fig. 1. Chemical structure of common PUFAs. (A): Linoleic acid (C18:2, u-6), (B): Alpha linolenic acid (C18:3, u-3), (C): Eicosapentaenoic acid (C20:5, u-3), (D): Docosahexaenoic acid
(C22:6, u-3) and (E): Arachidonic acid (C20:4, u 6) [Adapted from Holub (2002)].
the core (Gouin, 2004; Wang et al., 2014). In addition, microen- use in microencapsulation of various core materials, particularly
capsulation using complex coacervation can be carried out at mild PUFAs-rich oils, are the two foci of this review. Recent advances in
processing conditions without requiring toxic solvents (Gouin, complex coacervation science involve the preparation of complex
2004). Complex coacervates formed by the interaction of oppo- coacervates from plant protein and gum extracted from a single
sitely charged polymers are believed to be more surface active than plant and utilization of such plant protein-gum complex co-
the individual protein or polysaccharide; therefore, they are acervates to microencapsulate PUFAs-rich oil from the same plant.
regarded as better emulsifier and stabilizers in emulsion systems This review paper will also evaluate such complex coacervation
(Benichou, Aserin, Lutz, & Garti, 2007). This characteristic of com- techniques of microencapsulation. Digestion of oils and fats and
plex coacervates consequently helps to obtain increased encapsu- bioavailability of PUFAs are critical factors to be considered in food
lation efficiency and results into higher oxidative stability of the fortification and functional food development. Therefore, an
core. attempt has also been made to review relevant information on the
In the above context, this review mainly focuses and attempts to digestion of PUFAs in the physiological conditions.
provide a detailed overview of the complex coacervation method of Section 2 of this review provides an overview of the structural
microencapsulation that has been employed to stabilize PUFAs-rich organization of fatty acids and triacylglycerols and compares fatty
oils. Various aspects of microencapsulation of PUFAs-rich plant oils acid profiles of some important plant oils. Sections 3 and 4 provide
using complex coacervation technology have been reviewed else- a comprehensive overview of the important aspects of prevailing
where (Ducel, Richard, Saulnier, Popineau, & Boury, 2004). Com- microencapsulation technologies, principles and process of com-
plex coacervation of plant proteins and polysaccharides and their plex coacervation between proteins and polysaccharides and
372 Y.P. Timilsena et al. / Food Hydrocolloids 69 (2017) 369e381
application of resultant complex coacervates in encapsulating sn-1 position (Lei et al., 2012).
PUFAs-rich oils. Recent advancement in complex coacervation Plant seeds such as flax, chia, hemp, and perilla are rich in PUFAs
technology using plant protein-gum system is presented in Section (Table 1). The proportion of PUFAs in chia seed oil is higher than
5, whereas available literature on the physiological digestion pro- others listed in this table. The relative content of a-linolenic acid
cess involved in lipid metabolism is reviewed in Section 6. The final (>60%) and linoleic acid (~20%) is also superior to these oils. A
section (Section 7) provides concluding remarks and points to the comparison of the fatty acid profile of different plant seed oils
future direction/scope of complex coacervation research. (Table 1) indicates that chia seed oil provides a healthy balance of
two important essential fatty acids, i.e., alpha-linolenic acid and
2. Fatty acids and PUFAs profile and content in plant oils linoleic acid. The u-3 to u-6 fatty acid ratio of chia oils (3.18e4.18)
was also found to be higher than many vegetable oils (Alvarez-
Major lipids present in the plant oils are triacylglycerols Chavez, Valdivia-Lo pez, Aburto-Juarez, & Tecante, 2008). Chia
comprising more than 95% of the total oil mass (Singh, Ye, & Horne, seed oil was found to contain twelve species of triacylglycerols
2009). These triacylglycerols are comprised of three fatty acids which comprise of more than 90% of the oil. Trilinolenin was found
joined together onto a glycerol backbone through an ester linkage to be the prevalent triacylglycerol and a-linolenic acid was found to
(Fig. 2). Length of fatty acid chain, number and position of double present in most triacylglycerols of chia seed oil (Ixtaina et al., 2011;
bonds in them and positional distribution of fatty acids in the tri- Timilsena, Vongsvivut, Adhikari, & Adhikari, 2017).
acylglycerol molecule determine the physicochemical properties of
oils. 3. Complex coacervation process
Fatty acids are long molecules comprising a non-polar hydro-
carbon chain with a carboxyl acid group (COOH) at one end and a Coacervation is a term used in colloidal chemistry to denote the
methyl group (CH3) at the other end. The fatty acids can be satu- associative phase separation induced by altering the pH, ionic
rated, monounsaturated or polyunsaturated depending on the strength, temperature or solubility of the dissolving medium. If the
absence and presence of one or more double bonds (Kralovec, coacervation is induced in a single polymer system, it is known as
Zhang, Zhang, & Barrow, 2012). For example, oleic acid (OA) con- simple coacervation, while the one taking place between two or
tains one double bond at the ninth position from the methyl ter- more polymers is regarded as complex coacervation process. In
minus and docosahexaenoic acid (DHA) contains six double bonds complex coacervation process, electrostatic interaction takes place
with the first double bond occurring at the third carbon atom from between two oppositely charged polymer molecules and finally
the methyl terminus. Therefore, OA and DHA are known as u-9 and leads to phase separation. In many cases, the interaction between
u-3, respectively. protein and polysaccharide is used to produce complex coacervates
The composition of triglycerides is different in different fats and for food applications (Schmitt & Turgeon, 2011). Although elec-
oils. The triglyceride containing single type of fatty acid is known as trostatic force between oppositely charged macromolecules is the
monoacid triglyceride while the one containing different types of main driving force, van der Walls intermolecular forces and hy-
fatty acids is known as mixed triglycerides. A mixed triglyceride can drophobic interactions in proteins also affect the complex coacer-
exhibit different stereoisomeric forms depending on the type and vation process (Turgeon, Schmitt, & Sanchez, 2007).
position of fatty acid [a (outer) (sn-1 and sn-3) and b (middle) (sn- The coacervation process was systematically studied first by de
2) position] in the glycerol molecule. EPA and DHA in fish oil are Jong using gelatin and gum Arabic as reacting polymers (de Jong,
generally found in the sn-2 position, whereas they are present in 1949). Since then, a number of studies have been carried out to
the sn-1 or sn-3 positions in case of oils derived from marine better elucidate the complex coacervation process. However, the
mammals such as whales and seals (Genot, Meynier, Bernoud- mechanism of interaction between two biopolymers which leads to
Hubac, & Michalski, 2016; Wan, 2000). Thus positional distribu- the formation of the complex coacervates is so intricate and com-
tion of fatty acids in the triglyceride backbone not only provides plex that it is not yet adequately understood. It has been established
information about the origin of the oil but also important infor- that both the material properties such as molecular weight of
mation about the stability and bioavailability of the oil. For polymers, their charge density, concentration and mixing ratio and
example, fatty acids located at sn-2 position are reported to be process parameters such as method used for producing emulsion/
more resistant to autoxidation and exhibit superior bioavailability, degree of homogenisation, pH, temperature, and ionic strength
compared to those distributed in sn-1 or sn-3 position (Bandarra affect the complex coacervation process to a considerable extent
et al., 2016; Wang et al., 2015). It is also reported that saturated (Weinbreck, De Vries, Schrooyen, & De Kruif, 2003). Therefore, the
fatty acids and monounsaturated fatty acids are usually present in optimum condition for complex coacervation for one set of poly-
mers cannot be simply used for another set of polymers. Because of
this reason, the optimization of the coacervation parameters is the
essential priori step for its successful application in
microencapsulation.
Yield of complex coacervates as a function of dispersant pH and
polymer ratio is the most commonly used parameter for deter-
mining optimum condition for complex coacervation between two
polymers. This is because the coacervate yield can be easily deter-
mined by separating the precipitated complex coacervates from the
aqueous phase by centrifugation. Surface charge density (zeta po-
tential) and turbidity as a function of pH and polymer ratio are
recently and effectively used to optimise the complex coacervation
conditions between two biopolymers (Eratte, Wang, Dowling,
Barrow, & Adhikari, 2014; Timilsena, Wang, Adhikari, & Adhikari,
2016; Timilsena, Adhikari, Barrow, & Adhikari, 2016; Kaushik,
Fig. 2. (A): Structure of a triacylglycerol. R, R0 and R00 are the positions at which fatty Dowling, Barrow, & Adhikari, 2015; Wang et al., 2014). Surface
acids are linked (Adapted from Christie, 1986). electrostatic charge is the fundamental property of polymers. It is
Y.P. Timilsena et al. / Food Hydrocolloids 69 (2017) 369e381 373
Table 1
Monounsaturated (MUFAs) and polyunsaturated fatty acids (PUFAs) content of plant oils.a
Fatty Chia Flax Hemp Olive Sunflower Sesame Rapeseed Canola Walnut Coconut Perilla Peanut
acids
C16:1 0.2 0.09 0.11 1.8 0.12 0.11 0.21 e e e 0.07 0.07
(u-7)
C18:1 10.5 18.1 11.5 66.4 28.0 41.5 63.3 59.5 27.3 6.2 16.2 71.1
(u-9)
C18:2 20.4 15.3 59.4 16.4 62.2 40.9 19.6 18.8 50.0 1.6 17.9 18.2
(u-6)
C18:3 60.0 58.2 0.36 1.6 0.16 0.21 1.2 11.9 15.0 e 60.4 e
(u-3)
C20:1 0.2 0.2 16.5 0.30 0.18 0.32 9.1 e e e e e
(u-9)
C20:2 0.07 0.05 e e e e e e e e 0.05 e
Trans-fats e e e e e e 0.14 e e e e e
MUFAs 11.0 18.5 28.1 68.2 28.3 42.0 72.8 59.5 27.3 6.2 16.6 71.1
PUFAs 80.4 73.6 62.8 18.0 62.4 41.2 20.9 30.7 65.0 1.6 75.9 18.2
u-3 e e 0.4 1.6 0.4 0.2 1.2 e e 0.0 e 0.0
u-6 e e 62.4 16.4 62.2 40.9 19.6 e e 1.6 e 18.2
u-6: u-3 0.35 0.27 156.0 10.3 155.5 204.5 16.3 e e e 0.22 e
Reference Ciftci, Przybylski, Ciftci Orsavova Orsavova Orsavova Orsavova Orsavova Kostik, Dogan & Orsavova Osakabe et al., Orsavova
& Rudzin ska, 2012 et al., et al., et al., et al., 2015 et al., et al., Memeti, & Akgul et al., 2002; Ciftci et al., et al.,
2012 2015 2015 2015 2015 Bauer, 2013 (2005) 2015 2012 2015
a
Data are expressed as percentages of total fatty acid methyl esters (FAMEs).
determined by measuring electrophoretic mobility of biopolymer Ofner, 2004). However, due to toxic nature of glutaraldehyde, it is
molecules in a dispersant at a given temperature using combined not considered a suitable cross-linking agent for food application.
Doppler electrophoresis and phase analysis light scattering (Amin, Therefore, natural substances such as polyphenols and trans-
Rega, & Jankevics, 2012). In general, gums are negatively charged in glutaminase enzyme find increased application in food industries
a wide range of pH while proteins can be positively or negatively (Heck, Faccio, Richter, & Tho € ny-Meyer, 2013). The polyphenols
charged, depending on the pH of the medium. The optimum could form both covalent and non-covalent complexes with pro-
complexation is usually observed at a pH value where the polymers teins (e.g. zein), and their structural and functional properties
have opposite charges and the magnitude of charge difference depended on the nature of the complexes formed (Liu, Ma,
between the polymers is the highest (Liu, Elmer, Low, Nickerson, McClements, & Gao, 2016). On the other hand, transglutaminase
2010; Liu, Low Nickerson, 2010). Likewise, turbidity can be deter- catalyzes the formation of protein networks by introducing
mined by measuring the optical density or transmittance of a glutamyl-lysyl isopeptide bonds between target proteins (Fig. 4).
polymer-solvent mixture using UVeVis spectrophotometer. When
the complex coacervate particles (micro coacervates) start forming, 4. Microencapsulation of PUFAs-rich oil using complex
the turbidity of initially clear solution starts to increase. Once the coacervation
coacervation occurs to its highest degree the complex coacervates
start to coalescence giving rise to a visibly insoluble coacervate Microencapsulation of PUFAs-rich oils by simple emulsification
phase; hence two phase system is formed. This phase separation and spray drying (Shaw, McClements, & Decker, 2007), complexa-
stage of complex coacervation is known as macro-coacervation. The tion with proteins and polysaccharides (Augustin & Hemar, 2009)
denser phase or polymer-rich portion is known as coacervate phase and complex coacervation followed by spray drying (Eratte et al.,
and the lighter polymer deficient portion is called equilibrium 2014; Timilsena, Adhikari, et al., 2016) has successfully been car-
phase (Nairm, 1995) (Fig. 3 A). ried out. This process encapsulates the oils and fats in solid (pow-
Complex coacervation is a reversible process and the complex der) matrix. These solid forms of microcapsules can readily be
coacervates formed by the interaction of a protein and a poly- incorporated into other food and pharmaceutical formulations and
saccharide can dissociate when the conditions, especially the pH make the fortification process more effective.
and temperature are unfavourable. Complex coacervates are shelf- In general, complex coacervation-based microencapsulation
stable within a narrow range of pH, ionic strength and temperature. process consists of four steps. Firstly, aqueous solution of two
Therefore, careful monitoring of the process is essential to arrive at polymers is prepared. Protein solution is prepared at a temperature
optimum complex coacervation conditions and to produce stable beyond its gelling temperature and pH above its isoelectric point.
complex coacervates. Many complex coacervates, especially the Secondly, hydrophobic core (oil) is blended with the protein solu-
gelatin-based complex coacervates require further consolidation tion first and the mixture is homogenised in order to make fine oil-
when they are intended to be used as microencapsulating shell in-water (o/w) emulsion. Thirdly, aqueous solution of the other
materials (Timilsena, Wang, et al., 2016; Wang et al., 2014). Hence, biopolymer (polysaccharide, in general) is mixed with the o/w
complex coacervates are stabilised by either cross-linking or by the emulsion. If two biopolymers have optimum density of opposite
application of mild heat (Kamyshny & Magdassi, 2006). A number charges, complex coacervation occurs instantly and the complex
of synthetic and natural materials, including glutaraldehyde, tannic coacervates form a layer surrounding the oil droplets. If the bio-
acid, gallic acid and transglutaminase (Table 2), are used for cross- polymers do not have satisfactory level of opposite charge density,
linking the complex coacervates. Glutaraldehyde forms covalent adjustment of pH or temperature is required to induce opposite
binding between amino groups. This type of cross-linking is irre- charge density in order to initiate the formation of complex coac-
versible and the mechanical integrity of the cross-linked shell is ervate. These complex coacervates are usually insoluble in water.
enhanced in such a way that it can resist the effect of pH, tem- Adsorption or deposition of the insoluble coacervates on the sur-
perature and mechanical shear to a greater extent (Mwangi & face of the oil droplets leads to precipitation (phase separation) of
374 Y.P. Timilsena et al. / Food Hydrocolloids 69 (2017) 369e381
Fig. 3. Complex coacervation and phase separation process. (A): protein-protein complex coacervation (BSA: Bovine serum albumin, b-Lg: b-lactoglobulin and GB: gelatin B)
[Adapted from Pathak, Rawat, Aswal, & Bohidar, (2015), (B): Precipitation of the complex coacervate microcapsules of chia seed oil demonstrating distinct two phases [Adapted from
Timilsena, Adhikari, et al. (2016)].
Table 2
Cross-linkers used to consolidate the gelatin-based complex coacervates.
Abbreviations: MEE: Microencapsulation efficiency, FA: Formaldehyde, TG: Transglutaminase, GA: Glutaraldehyde, TA: Tannic acid.
Fig. 4. Protein crosslinking through transamidation reactions catalyzed by transglutaminase [Adapted from Heck et al. (2013)].
Y.P. Timilsena et al. / Food Hydrocolloids 69 (2017) 369e381 375
Wt Ws
MEE ¼ 100 % (1)
Wt
Wt
MEY ¼ 100 % (2)
Wi
Fig. 5. Multicore complex coacervate microparticles of tuna oil encapsulated into
where, MEE is microencapsulation efficiency (%), MEY is yield of the gelatin-sodium hexametaphosphate complex coacervates (Adapted from Wang et al.,
2014).
microcapsules (%), Wt and Ws are the mass (g) of total oil and
surface oil of the microcapsules, Wi is the mass (g) of oil introduced
into microcapsule and Wm is the mass (g) of the microcapsules coacervates as shell materials have significantly higher encapsula-
taken. tion efficiency and longer storage life compared to the solid mi-
Lipid oxidation produces hydroperoxides, ketones and alcohols crocapsules produced using either individual protein or gum as
are oxidation products. Therefore, the efficacy of microencapsula- shell material (Timilsena, Adhikari, et al., 2016). In most cases
tion is measured as delaying the onset of oxidation. Oxidative complex coacervation-based microencapsulation produces multi-
stability is usually measured at elevated temperature using Ranci- nuclear microcapsules surrounded by double layers of shell mate-
mat® or Oxypress®. In Rancimat® test, stability is measured as the rials, thus making it less porous and more robust. As a consequence,
time required to attain the inflection point of the conductivity curve it offers higher oxidative stability to PUFAs-rich oils than simple
indicated by the abrupt increase in the slope of the curve and is emulsification and drying process.
used as the oxidative stability index (OSI) (Timilsena, Adhikari,
et al., 2016). In ambient temperature, oxidative stability is
measured in terms of peroxide value, p-Anisidine value or Totox 5. Recent advances in complex coacervation
value (Totox ¼ p AVþ2(PV)). OSI is dependent on various factors
such as microcapsule shape, size, porosity and the surface oil of the Although the complex coacervation between various proteins
microcapsule. It has been found that higher the surface oil, lower is and opposite charged biopolymers such as polyphosphates and
the OSI. It is because the surface oil gets rapidly oxidised as it comes polysaccharides has been well reported, gelatin is probably so far
into the contact with air. the only commercialised protein for complex coacervation purpose
In general, microcapsules prepared using complex coacervates (Table 2) due to its excellent functional properties such as solubility,
as shell can be either mononucleated or multinucleated. The size emulsifying and gelling capability. At Ocean Nutrition Canada (now
and morphology of microcapsules can be highly affected by the DSM), commercial omega-3 oil powders with 60% oil loading, MEG-
processing conditions. Wang et al. (2014) demonstrated that mi- 3®, are currently produced with gelatin-sodium hexametaphos-
crocapsules produced through the complex coacervation between phate complex coacervates as the encapsulant (Yan & Zhang, 2014,
gelatin and sodium hexametaphosphate possess a core-shell chap. 12). In Wang et al. (2014)’s work, the preparation of this MEG-
structure in which each droplet of oil is covered by the thin layer 3® encapsulation matrix was systemically investigated and opti-
of shell and multiple core particles are surrounded by a second mised. The resulted fish oil microcapsule exhibited high oil loading
layer of shell producing a highly stable microcapsules of tuna oil (approx. 60%), low surface oil (<0.01%) and significantly improved
(Fig. 5). oxidative stability. As a result, MEG-3® has been successfully
Barrow & Shahidi (2007) reported that complex coacervation is incorporated into various food products including bakery, health
the most effective methods of microencapsulation of u-3 fatty acids supplements and infant formula. However, gelatin is relatively
and delivering them into foods. Timilsena, Adhikari, et al. (2016) expensive and cannot be consumed by the increasing vegetarian
demonstrated that microcapsules obtained by complex coacerva- and vegan population. Consequently, there is an increasing trend of
tion followed by spray drying possess higher encapsulation effi- plant protein use in the food industry, partly driven by their lower
ciency and increased oxidative stability than the microcapsules tendency to stimulate an allergic response compared to animal-
obtained by simple emulsification followed by spray drying. It derived alternatives (Choi et al., 2010). Hence, novel encapsula-
suggests that complex coacervation-based microencapsulation tion matrices based on complex coacervation technique require to
better protects PUFAs-rich oils than a simple spray drying. Higher be developed. The development of alternative protein-
encapsulation efficiency in complex coacervation-based microen- polysaccharide complex coacervates requires rigorous optimiza-
capsulation is resulted from the better surface active nature of the tion of the complex coacervation parameters because each pair of
complex coacervates. It has been reported that complex co- protein and polysaccharide form stable complex coacervates at
acervates migrate rapidly to the surface of the lipid droplets during specified pHs and biopolymer mixing ratios and the optimised
the emulsification stage and form a uniform layer around the oil conditions for one set of biopolymers cannot be replicated to
droplets (Benichou, Aserin, & Garti, 2002). These coacervates can be another set of biopolymers. Considerable research has been carried
converted into powder form either by freeze drying or spray drying. out in recent past to produce complex coacervates using milk and
The powder form of microcapsules produced using complex plant proteins such as whey, soy and pea proteins by optimizing
376 Y.P. Timilsena et al. / Food Hydrocolloids 69 (2017) 369e381
coacervation parameters (Table 3). These complex coacervates have under same test conditions. Similarly, the microencapsulated FSO
been utilised to microencapsulate u-3 oils. However, their stability was about 9 times more stable compared to the unencapsulated
and bioavailability of encapsulated oils have yet to be adequately one under same conditions. These two studies showed that plant
understood. This area of research is currently receiving increased protein-gum complex coacervates can be effectively used to
attention. encapsulate and stabilize PUFAs-rich oils.
Soybean protein and pea protein isolates are two of the most Characteristics of shell and core material are important factors
commonly used plant proteins as encapsulating shell materials determining encapsulation efficiency, core stability and controlled
(Ducel et al., 2004; Elmer, Karaca, Low, & Nickerson, 2011; Gai, Li, & release of the core. The bioavailability and bioefficiency of active
Jiang, 2012; Jun-xia, Hai-yan, & Jian, 2011; Klemmer, Waldner, substances also depend on the encapsulation and delivery system.
Stone, Low, & Nickerson, 2012; de Conto, Grosso, & Gonçalves, The design of an effective encapsulation system requires an un-
2013). Recently complex coacervates of lentil protein isolate (Aryee derstanding of mechanisms of encapsulation, chemical composi-
& Nickerson, 2012; Chang, Gupta, Timilsena, & Adhikari, 2016; tion of shell matrix and possible interaction of the active
Chang, Varankovich, & Nickerson, 2016) and canola protein compounds with the shell matrix (Augustin & Hemar, 2009).
isolate (Chang, Gupta, Timilsena, & Adhikari, 2016; Chang, Bioavailability refers to the fraction of an ingested nutrient that gets
Varankovich, & Nickerson, 2016) with chitosan or gum Arabic released at specified spots of alimentary canal and becomes avail-
were developed and used to encapsulate oils and other unstable able for absorption. As the digestion process is affected by the na-
and bioactive food ingredients (Table 4). These studies used protein ture of the food structure, the design of a suitable food matrix is
from one plant and polysaccharide from another to produce com- important to ascertain bioavailability of the nutrient. In order to
plex coacervates. There is a paucity of information on the formation maximize the benefits of PUFAs fundamental understanding of
of complex coacervates between proteins and gums from the same their release from microcapsule during their passage through
plant source. Until now, there are only two studies which report the gastrointestinal tract and subsequent absorption is vitally
science of formation of complex coacervates between protein and important.
gum of same plant species and subsequent application of these
unique complex coacervates in microencapsulation of PUFAs-rich
oils from the same plant (Kaushik et al., 2015, 2016; Timilsena, 6. Physiological digestion of encapsulated oils and fats
Wang, et al., 2016; Timilsena, Adhikari, et al., 2016). Kaushik et al.
(2015; 2016) studied the complex coacervation between flaxseed Majority of hitherto lipid digestion research (Mun, Decker, &
protein isolate (FPI) and flaxseed gum (FSG) and used such FPI-FSG McClements, 2007; Hur, Decker, & McClements, 2009; Sarkar,
complex coacervates as shell material to encapsulate PUFAs-rich Goh, Singh, & Singh, 2009; Sarkar, Horne, & Singh, 2010) has
flaxseed oil (FSO). Similarly, Timilsena, Wang, et al. (2016) and focussed studying the digestion of emulsified oil using a single or
Timilsena, Adhikari, et al. (2016) investigated the complex coacer- multi-compartment in vitro digestion model. These studies were
vation formation between chia seed protein isolate (CPI) and chia aimed at understanding the extent and nature of digestion of lipids
seed gum (CSG). The authors also used these CPI-CSG complex in each stage of the gastro-intestinal tract. Most of the reported
coacervates to microencapsulate PUFAs-rich chia seed oil. It can be studies deal with the digestion of oil-in-water emulsion systems. In
expected that this types of complex coacervation and microen- these emulsion systems, both the rate of lipolysis and their in vivo
capsulation system will provide better core-shell compatibility. In absorption are affected largely by the size of emulsion droplets and
both studies, the authors compared the effect of different core-to- the effective area available for lipase adsorption (Golding et al.,
shell ratio and different drying methods in the microencapsula- 2011). However, there are limited number of studies which inves-
tion efficiency and oxidative stability of the PUFAs-rich oils tigated the impact of individual microencapsulation technologies
(Table 4). It was shown that the microencapsulated CSO was at least on the digestibility and bioavailability of encapsulated oils and fats
6 times more stable against oxidation than the unencapsulated one (Augustin et al., 2014; Chung, Sanguansri, & Augustin, 2011). These
previous studies have been discussed in this section.
Table 3
Optimum pH and biopolymer ratio for the formation of complex coacervates between some biopolymers.
WPI Gum Arabic 3.0e4.5 4.0 2:1 Weinbreck, Minor, and De Kruif (2004)
SPI Gum Arabic 2.5e4.5 4.0 1:1 Jun-xia et al. (2011)
Chitosan Gum Arabic 2.0e4.0 3.6 4:1 Butstraen and Salaün (2014)
Gelatin CMC 9.0e11.0 9.0 1:1 Lii, Tomasik, Zaleska, Liaw, and Lai (2002)
Gelatin Chitosan 4.5e6.5 5.25e5.50 10:1e20:1 Remunan-Lopez and Bodmeier (1996)
Chitosan CMC 3.0e5.0 4.0 Tiyaboonchai & Ritthidej (2003)
SPI Pectin e 4.4 1:1 Mendanha et al. (2009)
LPI Gum Arabic 1.5e8.0 3.5 1:1 Aryee and Nickerson (2012)
PPI Gum Arabic 2.4e4.3 3.6 2:1 Liu, Elmer, et al. (2010) and Liu, Low, et al. (2010)
PPI Chitosan e 6.2 7.5:1 Elmer et al. (2011)
PPI Alginate 1.55e2.98 2.10 4:1 Klemmer et al. (2012)
Alpha gliadins Gum Arabic 2.0e4.0 2.75 3:7 Ducel et al. (2004)
Pea globulins Gum Arabic 2.0e4.0 3.0 1:1 Ducel et al. (2004)
BSA Pectin 1.6e4.7 4.7 5:1 Ru, Wang, Lee, Ding, and Huang (2012)
Gelatin Alginate 2.0e5.0 3.5e3.8 3.5:1 Shinde and Nagarsenker (2009)
b-lactoglobulin Gum Arabic 3.5e4.4 3.76 2:1 Sanchez, Mekhloufi, and Renard (2006)
Gelatin Pectin 3.8 1:1 Liu, Low, and Nickerson (2009)
Gum Arabic Chitosan 4.0e5.5 e 5.5:1 Espinosa-Andrews et al. (2013)
Note: CMC ¼ Carboxymethyl cellulose, SPI ¼ Soy protein isolate, PPI ¼ Pea protein isolate, BSA ¼ Bovine serum albumin, WPI ¼ Whey protein isolate, LPI ¼ Lentil protein
isolate.
Y.P. Timilsena et al. / Food Hydrocolloids 69 (2017) 369e381 377
Table 4
Microencapsulation of bioactive ingredients through complex coacervation of non-gelatin proteins and polysaccharides.
Polymer combinations Core Opt pH Biopolymer ratio MEE (%) OSI (h) Drying method References
WPI/GA Tuna oil 3.75 3:1 73 22.1 Spray & freeze drying Eratte et al. (2014)
SPI/GA Sweet orange oil 4.0 1:1 65e78 e Spray drying Jun-xia et al. (2011)
SPI/Pectin Casein hydrolysate 4.4 1:1 79e92 e Freeze drying Mendanha et al. (2009)
SPI/Pectin Propolis extract 4.0 e 66e72 e Freeze drying Nori et al. (2011)
CPI/CSG Chia seed oil 2.7 6:1 93.6 12.3 Spray & freeze drying Timilsena, Wang, et al. (2016) and
Timilsena, Adhikari, et al. (2016)
FPI/FSG Flaxseed oi 3.1 3:1 87.6 8.8 Spray & freeze drying Kaushik et al. (2015, 2016)
Abbreviations: MEE: Microencapsulation efficiency, OSI ¼ oxidative stability index, SPI: Soy protein isolate, GA: Gum Arabic, PPI: Pea protein isolate, GG: Guar gum, CPI¼ Chia
seed protein isolate, CSG ¼ Chia seed gum, FPI ¼ Flaxseed protein isolate, FSG ¼ Flaxseed gum.
Ilyasoglu and El (2014) studied the bioavailability of EPA and resistant to digestion and thus less oil is released in the gastric stage
DHA from sodium caseinateegum Arabic complex nanocapsules allowing more oil in the intact form to reach the intestinal stage.
and found that only 56.2% of EPA and 47.4% of DHA became avail- These authors also argued that main reason for slower digestion of
able after digestion of fish oil. These authors suggested that the oil encapsulated oil is because the oils/fats must be released from the
encapsulated in double-layered complex coacervate based nano- encapsulating matrix before it is acted upon by the enzyme (lipase).
capsules was only partially released and its digestion was greatly In addition, these shell materials affect the interfacial behaviour
reduced. A significant portion of oil from these nanocapsules was and interfere with the diffusion and adsorption of lipase to the oil
passed to the large intestine without digestion. Shen, Apriani, droplets. Presence of protein, fibres and polysaccharides in diges-
Weerakkody, Sanguansri, and Augustin (2011) also reported that tion mixture interfered lipase activity which reduced the rate of
only 78.6% of oil encapsulated into a composite matrix comprising digestion of lipid by pancreatic lipase (Mun et al., 2007). All the
sodium caseinate, glucose and HylonVII and incorporated into or- aforementioned studies suggest that physicochemical properties of
ange juice was digested, whereas extent of digestion was reduced microcapsules, such as their size, interfacial characteristics and
to 64% when it was incorporated into a cereal bar. This study pro- degree of crystallization of lipid significantly affect the rate and
vided a conclusive evidence that nature of food matrix influences extent of lipolysis in the gastro-intestinal tract (Augustin et al.,
the lipolysis process and that the presence of dietary fibres slows 2014).
down the lipolysis of the encapsulated oil. Patten, Augustin, Although a portion of the oil gets released from the microcap-
Sanguansri, Head, and Abeywardena (2009) studied in vivo di- sules in the oral stage, it passes to the stomach without digestion. In
gestibility of spray dried fish oil powders in mice and reported that human beings the actual digestion of lipids starts at stomach due to
only 4e6% of the oil was released from the microcapsules and small the action of gastric lipase. Gastric lipase being active only at a pH
quantity of low chain PUFAs were present in the plasma indicating range between 4.0 and 6.0, it remains inactive at pH values of the
that large quantity of oil reached the bowel without being digested normal conditions of stomach, where the pH normally lies between
in the small intestine. 1.5 and 3.0. However, the pH of human stomach increases upto the
Augustin et al. (2014) compared the digestion behaviour of 12 optimum value for gastric lipase due to buffering effect of foods. It
types of spray dried powders of canola oil-in-water emulsion and has been reported that only 10e30% of lipids is digested in the
reported that lipolysis of canola oil in these powders varied be- gastric phase and produces a mixture of free fatty acids, diac-
tween 12 and 68%. This study suggested that in vitro digestibility of ylglycerols (DAGs) and monoacylglycerols (MAGs) (Bauer, Jakob, &
oil encapsulated in powders depends on both formulation and the Mosenthin, 2005; Gallier & Singh, 2012). After the completion of
processing steps used in their manufacture. Then, these authors the gastric digestion, the oil along with other food (the partially
selected 5 different formulations of oil-containing microcapsule digested food is known as chyme) enters into the small intestine
powders and incorporated them into a dairy beverage intended for where it is mixed with enzymes (pancreatic lipase, a-amylase and
in vivo human trial. Free or unencapsulated oil incorporated into proteases-trypsin and chymotrypsin), salts (bicarbonates and bile
the same beverage was used as the control. They found that tri- salts) and electrolytes (sodium and calcium). The acids present in
glyceride levels in blood of the subjects fed with the micro- the chyme is neutralised by bicarbonates so that the pH of the in-
encapsulated oil was higher than the subjects fed with testinal content generally reach up to 7.5. Majority of the lipids
unencapsulated oil. Most in vitro digestion studies of lipids have (70e90% of the lipids) present in foods get digested in the small
focussed on quantifying the release of the oil and the production of intestine by the action of pancreatic lipases (Wickham, Wilde, &
free fatty acids as an indicator of the lipolysis. The effect of non-lipid Fillery-Travis, 2002). The ultimate products of lipid digestion are
ingredients in slowing down the digestion of lipids has not been monoglycerides and fatty acids (Fig. 6). Lipid digestion and ab-
studied and explained to date. Comparison or validation of data sorption is a remarkably efficient process with more than 95% ef-
obtained from in vitro studies with in vivo experiments is another ficiency and there is no feed-back regulation to reduce its
challenge that has not been covered so far. assimilation (Patton et al., 1985). The products of lipid digestion are
Timilsena, Adhikari, Barrow, & Adhikari (2017) and Timilsena, absorbed through the epithelial cells and carried into the blood
Vongsvivut, et al. (2017) reported that pattern and kinetics of stream (Seidel & Long, 2006).
digestion of unencapsulated oils is quite different than the diges- During lipid digestion, it is very important that oil droplets
tion of the encapsulated oil. These authors also showed that remain emulsified in the stomach and intestinal fluids so that they
digestion of encapsulated oil is influenced by the nature of the shell become accessible to the enzymes (e.g. gastric and pancreatic li-
materials in which they are encapsulated. Protein-based matrices pases). Emulsification is very essential in order to bring the hy-
get readily digested in the stomach releasing the oil from the ma- drophobic (lipid) and hydrophilic (lipase) components together.
trix, whereas gum-based matrices are not digestible but get solu- The water soluble lipase has to be able to attach at the lipid (oil)-
bilised in the stomach and intestinal conditions. On the other hand, water interface for lipolysis to occur. Therefore, any factor that af-
matrices prepared from the protein-gum complexation are more fects the migration of lipases to oil-water interface also affects the
378 Y.P. Timilsena et al. / Food Hydrocolloids 69 (2017) 369e381
Fig. 6. Breakdown of triacylglycerols (TAGs) into a monoacylglycerol (MAG) and two free fatty acids (FFAs) molecules.
lipid digestion process (Singh & Gallier, 2014). Emulsification in the oxidative deterioration unless any protective measures are applied.
stomach leads to the organization of dietary lipids in the form of Oxidation of PUFAs-rich oils not only lowers their nutritional value
droplets in the aqueous digestive system (Bauer et al., 2005). Size of and sensory attributes but also generates toxic substances. There-
the emulsified lipid droplets is also very important factor affecting fore, it is essential to innovate and adopt a suitable microencap-
lipid digestion. The smaller the droplet, the larger is the surface sulation technology to maximize the health benefits of PUFAs. In
area available for lipase adsorption and faster is the lipid digestion. general, they are encapsulated into edible and oxygen resistant
The nature of emulsifier and the molecular structure of tri- food matrices. For this purpose, complex coacervation based
acylglycerol molecules such as the chain length and degree of microencapsulation is more preferable because of substantially
saturation of the fatty acids and their position on the triacylglycerol high microencapsulation efficiency and increased oxidative stabil-
backbone also influence the digestion of lipids (Gallier & Singh, ity achievable in this method even at high payload.
2012; Singh & Gallier, 2014). Although fish oil is rich in PUFAs, the demand for plant based
Lipid digestion in isolated conditions is quite different than that omega-3 rich oils is increasing due to the healthy image of the latter.
in the presence of non-lipid components such as proteins, poly- Similarly, despite high effectiveness of gelatin to encapsulate
saccharides and fibres. It has been reported that there is large in- PUFAs-rich oils, there is continued search for its alternative, pref-
fluence of these non-lipid components in the degree and the erably a plant protein due to the demand from vegetarian and vegan
kinetics of lipid digestion and bioavailability (Augustin et al., 2014; population. Recent trends are to utilize plant protein-gum complex
Singh et al., 2009). Better understanding of the mechanisms of lipid coacervates in stabilization of PUFAs-rich oils and other sensitive
digestion and factors affecting its degree and kinetics will enable bioactive food ingredients. Microencapsulation of chia seed oil and
better design of encapsulating shell materials so that release and flaxseed oil using protein-gum complex coacervates produced from
physiological digestion of lipids can be controlled according to the same plant species has shown better compatibility between the
bodily requirement, eventually reducing the risk of diseases that core and the shell. Controlled release of PUFAs and their subsequent
result from higher intake of fats (Bornhorst & Paul Singh, 2014; digestion in the gastro-intestinal tract are important consideration
Sarkar et al., 2010; Singh et al., 2009; Zhang, Zhang, Zhang, for successful design of PUFAs-rich microcapsules. However, there is
Decker, & McClements, 2015; Zhu, Ye, Verrier, & Singh, 2013). In acutely limited information on the digestion of microencapsulated
practice, food digestion studies are carried out using a relatively oils. In vitro studies followed by in vivo trials would more accurately
simpler in vitro models and more complex in vivo animal and hu- evaluate the effectiveness of microcapsules and microencapsulation
man trials. During in vitro experiments, food is dispersed in simu- technologies applied to PUFAs in protecting them from degradation
lated digestive fluids and mechanical movement and the digestion and controlling their bioavailability in the body.
of targeted substance over time is measured. Although it is very Co-encapsulation of two or more bioactive food ingredients in a
difficult to mimic the complexity of real human digestion system, single matrix is a recently introduced concept in food industries.
nevertheless these in vitro models provide insights into the Wang et al. (2015) reported a successful co-encapsulation tech-
fundamental mechanisms of detachment of the bioactive food in- nology of omega-3 oil with vitamin A, D3, E, K2, curcumin and co-
gredients from the complex food matrix and their subsequent hy- enzyme Q10 in a gelatin-sodium hexametaphosphate complex
drolysis in the gastro-intestinal environment (Chung et al., 2011; coacervate. Eratte et al. (2015) also reported co-encapsulation of
Mackie, 2012, pp. 49e70). Besides, these in vitro models allow in- omega-3 rich oil with probiotic bacteria in whey protein isolate-
dependent study of many factors which cannot be done in in vivo. gum Arabic complex coacervate shells. In both these studies, the
Furthermore, in vitro studies do not require stringent ethical authors used animal derived proteins. Replacing omega-3 rich fish
approval; thus they can be carried out at much reduced cost (Kong oils with plant oils and replacing animal-based proteins (gelatin,
& Singh, 2009). Due to these reasons, in vitro experiments are still whey protein) with plant proteins and application of the co-
popularly used in digestion studies before proceeding to the full- encapsulation concept to more than one active functional in-
fledged in vitro trials. Ultimately, the results obtained from gredients can confer multiple benefits and could be important
in vitro models have to be validated using data obtained from in vivo future research direction.
animal and/or human trials (Bornhorst & Paul Singh, 2014).
Acknowledgement
7. Concluding remarks and future perspectives
The first author gratefully acknowledges the scholarship sup-
Although PUFAs-rich oils contribute significantly in the health port from RMIT University, Melbourne, Australia and CSIRO,
and wellbeing of consumers, they are highly susceptible to Australia.
Y.P. Timilsena et al. / Food Hydrocolloids 69 (2017) 369e381 379
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