Koupantsis_Flavour encapsulation in milk protein and CMC complex coacervate_2014

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Food Hydrocolloids 37 (2014) 134e142
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Food Hydrocolloids
journal homepage: www.elsevier .com/locate/ foodhyd
Flavour encapsulation in milk proteins e CMC coacervate-type
complexes
T. Koupantsis a, E. Pavlidou b, A. Paraskevopoulou a,*
a Laboratory of Food Chemistry and Technology, School of Chemistry, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece
b Solid State Physics Section, Physics Department, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece
a r t i c l e i n f o
Article history:
Received 2 July 2013
Accepted 31 October 2013
Keywords:
Flavour encapsulation
Complex coacervation
Sodium caseinate
Whey protein
Carboxymethylcellulose
Response surface methodology
* Corresponding author. Tel.: þ30 2 31 0 997832; fa
E-mail address: adparask@chem.auth.gr (A. Parask
0268-005X/$ e see front matter � 2013 Elsevier Ltd.
http://dx.doi.org/10.1016/j.foodhyd.2013.10.031
a b s t r a c t
Beta-pinene containing microcapsules were prepared by complex coacervation of milk proteins, i.e so-
dium caseinate (CN) and whey protein isolate (WPI), with carboxymethylcellulose (CMC). Milk proteins
e CMC interactions were followed by z-potential measurements, while the initial emulsions were
characterised for droplet size and biopolymer amount present at the oil/water interface. Response sur-
face methodology was applied to investigate the effects of encapsulation processing variables, including
protein/polysaccharide (pr/pl) ratio and volatile compound’s mass, on encapsulation yield, loading and
efficiency as well as the morphological characteristics of the produced microcapsules. The obtained
results revealed that it was possible to encapsulate b-pinene with milk proteins and CMC by complex
coacervation, while most of the characteristics evaluated were affected by the process variables. Co-
acervates prepared at the highest pr/pl ratio of 6.99 and b-pinene mass (6.99 g) were the most effective
in encapsulating the flavour compound, something that was more evident in the case of WPIeCMC
mixture. Additionally, microcapsule structure, evaluated by Scanning Electron Microscopy analysis, was
noticeably affected by the protein/polysaccharide ratio being compact when pr/pl was low and “spongy”-
like when it was high.
� 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Proteinepolysaccharide interactions have found diverse appli-
cations in food sector, such as in food colloidal systems formation,
fat substitution, protein recovery from fluid by-products and
encapsulation (Damianou & Kiosseoglou, 2006; Dickinson, 2003;
Kika, Korlos, & Kiosseoglou, 2007; Paraskevopoulou &
Kiosseoglou, 2013). Among them, biopolymer-based encapsula-
tion of various ingredients (flavours, vitamins, antioxidants, etc.)
offers many advantages in view of protection from losses and
environmental factors, unpleasant tastes’ masking, liquid conver-
sion to more convenient solid materials and controlled release
(Nori et al., 2011; Qv, Zeng, & Jiang, 2011; Zhang, Pan, & Chung,
2011). In the case of flavour compounds, encapsulation provides
an effective way for their stability reinforcement through elimi-
nation of problems associated with their exposure to air, light, heat
and moisture. It is based on the formation of a coating layer (wall
material) to encapsulate flavour droplets or particles (core mate-
rial). Proteinepolysaccharide complex coacervation is the process
x: þ30 231 0 997847, 997779.
evopoulou).
All rights reserved.
most commonly used for this purpose. It is, almost entirely,
accomplished when two oppositely charged biopolymer molecules
are mixed at a pH below the protein isoelectric point (pI) leading to
the formation of a complex, i.e. coacervate, which precipitates
retaining at the same time the aroma material at the coacervate
phase. The success of the process is mainly affected by pH manip-
ulation. More specifically, a soluble proteinepolysaccharide com-
plex is firstly formed followed by complex coacervation and/or
precipitation upon further lowering of pH, attributed to the mod-
ulation of initial repulsive proteinepolysaccharide interactions into
net attractive ones (Paraskevopoulou & Kiosseoglou, 2013).
Flavour encapsulation by complex coacervation of proteins with a
number of charge-carrying polysaccharides has been the subject of
some recently published research papers (Jun-xia, Hai-yan, & Jian,
2011; Leclercq, Harlander, & Reineccius, 2009; Prata, Zanin, Ré, &
Grosso, 2008; Yeo, Bellas, Firestone, Langer, & Kohane, 2005). In these
studies, various combinations including gelatine or soybean protein
isolate with gum arabic, xanthan or pectin have been used for the
encapsulation of essential oils or individual flavour compounds.
In the present study milk proteins, i.e. sodium caseinate and
whey protein isolate, were employed in admixture with carboxy-
methylcellulose. Milk proteins are commonly used in the food in-
dustry for their excellent functional (emulsion preparation and
Delta:1_given name
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T. Koupantsis et al. / Food Hydrocolloids 37 (2014) 134e142 135
stabilisation, water and fat binding, thickening, gelation) and
nutritional properties. CN is composed of a mixture of four phos-
phoproteins, namely as1-, as2-, b- and k-casein. Their highly disor-
dered and hydrophobic character is considered to be responsible
for sodium caseinate’s rapid absorption to the oilewater interface
during emulsification as well as stabilisation of oil droplets against
flocculation and coalescence (Dickinson, 1997). The major compo-
nent of WPI is b-lactoglobulin, a globular protein known to interact
with many aroma compounds due to the existence on the molecule
of two independent binding sites (Narayan & Berliner, 1997).
Carboxymethylcellulose was selected because it is an anionic
polysaccharide that is already widely used as a functional ingre-
dient by the food industry. Its manufacture involves treating cel-
lulose with aqueous sodium hydroxide followed by reaction with
monochloroacetic acid. It is water soluble and its solutions exhibit
non-Newtonian, pseudoplastic solutions (Zecher & Gerrish, 1999).
Milk proteins have been successfully used in combination with
polysaccharides such as gum arabic, xanthan, mesquite gum as well
as CMC in the formation and stabilisation of food emulsion systems,
while, depending on pH, both of them interact with poly-
saccharides to form soluble or insoluble complex coacervates,
nanoparticles or precipitates (Bedie, Turgeon, & Makhlouf, 2008;
Benichou, Aserin, Lutz, & Garti, 2007; Ercelebi & Ibanoglu, 2007;
Koupantsis & Kiosseoglou, 2009; Liu et al., 2012; Weinbreck,
Minor, & de Kruif, 2004; Ye, 2008). More specifically, it was
observed that when the pH of an aqueous WPIeCMC solution was
steadily reduced to values between 3.5 and 4, a gel-like systemwas
generated upon ageing (Koupantsis & Kiosseoglou, 2009;
Paraskevopoulou, Tsioga, Biliaderis, & Kiosseoglou, 2013). The
development of the gel structure was primarily attributed to elec-
trostatic interactions between the two biopolymers, while hydro-
phobic interactions between protein molecules appeared to play a
minor role. Additionally, the gel structure formation at low pH
enhanced the retention of orange oil aroma compounds when
compared with single biopolymers or their mixtures at neutral pH
(Paraskevopoulou et al., 2013).
In view of the above, this work was conducted to investigate
whether complex coacervation of milk proteins with CMC can be
used for the encapsulation of flavour compounds, i.e. b-pinene. The
initial emulsions were characterised for droplet size and
biopolymer adsorptionto the oil/water interface, while the mi-
crocapsules were characterised for encapsulation efficiency,
loading, yield and morphology. Optimisation of the encapsulation
process conditions was attempted using response surface meth-
odology where the simultaneous effect of b-pinene amount and
protein/polysaccharide mass ratio was investigated.
2. Materials and methods
2.1. Materials
Casein sodium salt (from bovine milk) was purchased from
Sigma Chemical Co. (Germany). Commercial whey protein isolate
(natural, unflavoured, lecithin content 95%, CAS No.127-91-3) were purchased from Fluka
(Switzerland). Analytical grade n-hexane was obtained from Merck
(Germany). Sodium azide (Aldrich, Germany) was added to the
protein and polysaccharide solutions to prevent microbial growth
(final concentration of 0.01% w/v). Hydrochloric acid and sodium
hydroxide solutions used for pH adjustment were purchased from
Aldrich (Germany). Deionised water was used for the preparation
of all solutions and emulsions.
2.2. Microcapsule preparation
2.2.1. Preparation of biopolymer matrices
Individual protein (3.5% w/v) and polysaccharide (0.5% w/v)
matrices were initially prepared by dispersing sodium caseinate,
whey protein isolate and carboxymethylcellulose in deionised
water under mechanical stirring at room temperature for more
than 5 h for a full dispersion of the macromolecules. The three
solutions were then used to prepare the emulsions. Blends con-
taining 0.25% (w/v) CMC and different amounts of CN or WPI were
obtained by mechanical mixing at 300 rpm (IKA, Malaysia) for at
least 30 min to assure the complete formation of biopolymer
mixtures (Table 1).
2.2.2. Formation of o/w emulsions
Oil-in-water emulsions were prepared at room temperature by
mixing appropriate amount of b-pinene with 200 mL protein/poly-
saccharide solution with the aid of a mechanical stirrer at 600 rpm
(IKA,Malaysia), followed by homogenisation at 30�106 Pa (4 passes)
using an APV-2000 pressure homogenizer (APV Systems, Denmark).
The produced emulsions showed afinal composition,which is shown
in Table 1. Sodium azide (0.01% w/v) was added to all emulsions in
order to prevent microbial growth. The losses during emulsion
preparationwere found tobevery limited (0.1 g of the
freeze-dried powder in a screw-capped glass tube, followed by
2 mL hexane (pH was adjusted to 7.0 by using a 0.1 mol/L sodium
hydroxide solution). The resulting solution was vortexed for 1 min
at room temperature and stored at �18 �C overnight. Immediately
after it was taken out of the freeze, it was centrifuged at 4000 rpm
for 40 min, placed in a water bath of 50 �C for 40 min and re-
centrifuged at 4000 rpm for 10 min until complete phase separa-
tion. The organic phase was collected and was subsequently stored
in screw-capped glass vials at �18 �C until further analysis. Each
powdered sample was extracted in triplicate.
2.7. GC-FID analyses
The analyses were accomplished with an Agilent 6890A gas
chromatograph equipped with a split-splitless injector and a
flame ionization detector (FID). The samples were analysed on a
HP-FFAP column (25 m � 0.20 mm i.d., film thickness 0.30 mm;
Agilent Technologies). The carrier gas was helium at a constant
flow rate of 1 mL/min. Samples (2 mL) were injected manually into
the GC in split mode with 1:100 ratio. Injector and detector were
both kept at 230 �C. The temperature programwas 40 �C for 2 min,
raised to 100 �C at 10 �C/min, then raised to 230 �C at 30 �C/min
and held for 1 min. The obtained peak areas were converted to
concentrations using a calibration curve (y ¼ 0.3732x þ 44.656).
For its construction, b-pinene solutions were prepared in hexane
at ten different concentration levels (0.10e6.24 g/L) and analysed
five times applying the same conditions as described previously
for the samples. Linear correlation coefficient was found equal to
0.9985. All analyses were performed at least three times
(CVof b-pinene-containing microcapsules production
was standardised for the maximum encapsulation by using
response surface methodology. As has already been mentioned,
two different biopolymer combinations were tested, i.e. CMC was
combined either with CN or WPI by using complex coacervation.
Three parameters were applied for the characterisation of dried
microcapsules, namely yield of the encapsulation procedure (EY),
encapsulation loading of b-pinene (EL) in the final product and
encapsulation efficiency (EE), as well as fresh emulsion droplet size
and biopolymer surface amount. Their values, generated from the
thirteen experimental runs, are provided in Table 1.
As statistical analysis revealed there was no evidence of “lack-
of-fit” since p values of all parameters were higher than 0.05
(Table 2). Moreover, the coefficient of determination was found to
be within 0.8272 and 0.9995 (mean w 0.9517) confirming the
desirability of the model to elucidate the relationships between the
variables thus allowing an acceptable fitness of response surface
models to the experimental data. Moreover, a high value for R2 (adj)
demonstrates that non-significant terms have not been included in
themodel (data not shown). The above results clearly show that the
chosen model can satisfactory explain the effect of the two factors,
i.e. proteinepolysaccharidemass ratio (A) and b-pinenemass (B) on
response parameters, i.e. EY, EL, EE, Gs, D[3,2].
Table 2
Regression coefficients and their p values from analysis of variance for the response vari
Responsea EY EE EL
Term Coef. p Coef. p Coef.
CNeCMC Const. 26.126 0.000** 20.052 0.000** 11.380
Ab 2.932 0.004** �2.897 0.004** �4.236
B �8.596 0.000** 1.666 0.046* 6.502
A2 �2.552 0.011* 1.957 0.033* 3.063
B2 4.836 0.000** 3.312 0.003** �0.053
A*B �0.853 0.413 2.223 0.413 0.115
Lack of fit 0.091 0.240
R2 0.9709 0.8834 0.9649
WPIeCMC Const. 21.890 0.000** 26.376 0.000** 15.726
A �2.761 0.003** 0.048 0.969 �4.233
B �7.695 0.000** 5.477 0.002** 7.148
A2 �9.526 0.011* 2.780 0.064 2.424
B2 4.279 0.000** �2.315 0.109 �1.886
A*B 1.223 0.213 2.610 0.161 2.260
Lack of fit 0.090 0.114
R2 0.9842 0.8272 0.9087
* For pFigs. 2b and 3b show that the
higher the core material concentration and the lower the pro-
tein:CMC ratio, the higher the encapsulation process loading.
T. Koupantsis et al. / Food Hydrocolloids 37 (2014) 134e142140
Probably, low protein content allows for a more satisfactory asso-
ciation between protein and CMC at the interface resulting in the
formation of a more “structured” interfacial film than higher pro-
tein content.
Both wall material concentration as well as core material mass
also affects the process in terms of encapsulation efficiency. As
Table 1 revealed, EE ranged from 17.8 to 29.02% and 13.05 to 35.54%
in the case of CNeCMC and WPIeCMC, respectively. The statistical
analysis suggested that the model was appropriate with satisfac-
tory R2 (0.8834 and 0.8272) and no evidence of “lack of fit” since for
CNeCMC and WPIeCMC p values were 0.240 and 0.114 (>0.05),
respectively (Table 2). Sodium caseinateeCMC microcapsules for-
mation was found to be considerably affected by the mass of core
material as well as the biopolymer content. More specifically, EE
was significantly influenced by the negative linear effect of A, the
positive linear effect of B and the positive quadratic effects of both
factors (Table 2, Fig. 2c). This last effect of A and B can be seen by the
characteristic curvature of the response surface, especially at values
near the middle level of both factors (Fig. 2c). The microcapsules
made using biopolymer mass and core material of 3.60 had the
lowest encapsulation efficiency (w20%), indicating that relatively
greater amount of b-pinene remained in the supernatant instead of
being encapsulated in the microcapsules at this ratio. It could be
hypothesised that the increase of biopolymer ratio (i.e. protein
concentration) induced the development of either proteinepoly-
saccharide or proteineprotein interactions reducing thereby the
ability of protein molecules to reach and become adsorbed at the
oil-water interface. In the case of WPIeCMC microcapsules,
encapsulation efficiency was appreciably affected by the b-pinene
content (positive linear effect at p > 0.05), i.e. EE was increased when b-pinene content
increased. Furthermore, as results of both Tables 1 and 2 revealed,
the amount of protein adsorbed on the oil droplet surface (Gs) was
greatly decreased by b-pinene mass increase (at p 0.05). It seems likely that the droplets present in emulsions
prepared with high b-pinene addition were not fully covered by
biopolymer molecules than those in emulsions prepared with low
b-pinene content. This observation is also connected to droplet size
increase (positive linear effect of B on D[3,2]) allowing inferring that
the increase in the concentration of core material enhanced in-
teractions between the biopolymers used resulting in increased
encapsulation efficiency. In general, our results are lower than
those reported in the literature something that partially could be
attributed to some extent to the fact that no cross-linking agent was
used. For example, the efficiency of the encapsulation of propolis by
soy protein and pectin as well as capsaicin encapsulation by
gelatine-Tween 60 mixture reached a 70% (Nori et al., 2011; Wang,
Chen, & Xu, 2008). In addition, the method applied for capsules
drying, i.e. freeze-drying, may induced losses of the volatile con-
stituent possibly through capsule disruption at very low
temperatures.
The encapsulation efficiency depicts the potency of biopolymers
firstly in the emulsification and stabilisation of volatile’s droplets
and secondly in the protection of the encapsulated aroma com-
pound against losses or oxidation, something that is very important
in the food industry for volatile core materials. As the mean droplet
size of emulsions was practically the same (D[3,2] ¼ 0.290 and
0.319 mm for CNeCMC and WPIeCMC systems, respectively), the
results obtained could be attributed to the differences between the
biopolymer materials used and their physicochemical properties
(e.g. surface activity, tendency to bind aroma compounds). WPI in
combinationwith CMC appeared to be rather more effective for the
encapsulation of b-pinene than sodium caseinate (Figs. 2c and 3c;
Table 1). Other studies have also shown that whey protein was
more effective in binding aroma compounds in comparison to so-
dium caseinate (Hansen & Booker, 1996; Li, Grün, & Fernando,
2000). A similar trend was also observed by Paraskevopoulou,
Tsoukala, and Kiosseoglou (2009) who noted that WPI was more
effective than CN and Tween 40 in retaining the mastic gum oil
volatiles (mainly terpenes) in hydroalcoholic model emulsion sys-
tems. The decrease in volatility of the hydrophobic terpenes was
attributed to hydrophobic interaction with the central cavity of the
protein andmore specifically with b-lactoglobulin (b-lg). According
to Guichard and Langourieux (2000), the b-lg molecules interact
with several aroma compounds (i.e. carbonyl compounds, ionones,
hydrocarbons) through two different binding sites (Narayan &
Berliner, 1997). Acidic pHs favour these interactions by enhancing
the flexibility of the protein molecule and assisting in molecule
unfolding and exposure of hidden residues to the surface (Jouenne
& Crouzet, 2000). Additionally, as was stated by Paraskevopoulou
et al. (2013), the relative amount of the terpenes in the headspace
of CMCeWPI-containing systems was significantly lower than in
the water phase at both pH values (6.0 and 3.7) studied probably
due to hydrophobic interactions with the system components.
Upon gelation at pH 3.7, the retention degree was further increased
as a result of the entrapment of the aroma compounds inside the
developed gel network.
3.4. Morphology of microcapsules
The morphology of the produced microcapsules was studied by
SEM. The images obtained revealed that the final freeze-dried
products exhibited either a compact or a “spongy”-like structure
(Fig. 4), which is common for this type of products as stated in the
literature (Quispe-Condori, Saldaña, & Temelli, 2011; Saravanan &
Panduranga, 2010). In general, the porosity of these structures ap-
pears to be influenced by both the biopolymer and b-pinene con-
centration. Similar findings have been reported by other
researchers by using another observation technique, such as X-ray
microscope (Laine et al., 2010). In both proteinepolysaccharide
combinations, i.e. CNeCMC and WPIeCMC, the surface of freeze
dried products with low protein concentration (Fig. 4a and d)
exhibited the lower porosity with the droplets coming closer to
each other and leaving less space between them, with the second
being the denser and more rigid. By increasing the protein con-
centration, as Fig. 4b for CN and 4e for WPI show, a network was
formed inwhich b-pinene droplets were immobilized. The network
of sodium caseinate was formed by chains with rounded edges
while that of WPI was flatter with sharper edges. The formation of
this network was the result of electrostatic interactions between
non-adsorbed proteins and polysaccharides molecules. Further-
more, z-potential values (Fig. 1a and c) corresponding to pH around
2.8 favour electrostatic interactions and bridging flocculation
(Dickinson, 2003). In the case of systems that contained the lowest
amount of b-pinene (Fig. 4c and f), the same network consisted of
elongated chains with branches seemed to be formed.
By comparing percentages of encapsulation efficiency (Figs. 2c
and 3c) and loading (Figs. 2b and 3b) with the morphology char-
acteristics of the final freeze-dried products it was concluded that
both efficiency and loading seemed to be affected by morphology.
More specifically, the “spongy”-like structures exhibited lower
percentages of encapsulation loading and efficiency than the
compact structures. The empty space of “spongy”-like structures
resulted indroplets of a larger free surface, thus increasing losses of
the volatile constituent. In all cases, the surfaces of the freeze-dried
products were smooth and without dents or shrinkages. In general,
the microstructure of the final product plays an important role and
the presence of cracks and dents on the surface of the dried
Fig. 4. SEM images of powders produced from different CNeCMC ratios (aec) and b-pinene mass (def): (a) CN/pl ratio 0.2 & b-pinene mass 6.9 g (�1000), (b) CN/pl ratio 6.9 & b-
pinene mass 6.9 (�1500), (c) CN/pl ratio 6.9 & b-pinene mass 0.2 (�1000), (d) WPI/pl ratio 0.2 & b-pinene mass 6.9 g (�1000), (e) WPI/pl ratio 6.9 & b-pinene mass 6.9 (�1000) and
(f) WPI/pl ratio 6.9 & b-pinene mass 0.2 (�1000).
T. Koupantsis et al. / Food Hydrocolloids 37 (2014) 134e142 141
particles affects the retention or losses of aroma compounds
(Druaux & Voilley, 1997).
4. Conclusions
The lowering of pH to a value near 2.8 of aqueous CNeCMC and
WPIeCMC mixtures resulted in the development of coacervate-
type complexes that can act as encapsulating agents. As results
revealed, most of the produced coacervates could be effective
encapsulation systems as well as possible delivery systems in foods.
This was more evident in the case of WPIeCMC mixture, possibly
due to the enhancement of the protein flexibility and unfolding at
acidic pH. In general, protein/polysaccharide ratio increase caused
changes in microcapsule morphology, producing a network in
which b-pinene droplets were immobilized. The formation of this
network was the result of electrostatic interactions between non-
adsorbed proteins and polysaccharides molecules. The use of
crosslinking agents may improve encapsulation efficiency of milk
proteinseCMC coacervates something that is under investigation
by our team.
Acknowledgements
The authors wish to thank Dr. F. Mantzouridou for advice
regarding the application of response surface methodology as well
as Dr. C. Malhiac and Dr. N. Hucher (University of Le Havre, France)
for their help in the determination of z-potential.
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	Flavour encapsulation in milk proteins – CMC coacervate-type complexes
	1 Introduction
	2 Materials and methods
	2.1 Materials
	2.2 Microcapsule preparation
	2.2.1 Preparation of biopolymer matrices
	2.2.2 Formation of o/w emulsions
	2.2.3 Preparation of microcapsules
	2.3 Emulsion droplet size measurement
	2.4 ζ-Potential measurements of protein and polysaccharide solutions
	2.5 Protein and polysaccharide surface amount determination
	2.6 Extraction of encapsulated β-pinene
	2.7 GC-FID analyses
	2.8 Microcapsules characterisation
	2.9 Experimental design
	3 Results and discussion
	3.1 pH selection
	3.2 Response surface methodology
	3.3 Encapsulation yield, loading and efficiency
	3.4 Morphology of microcapsules
	4 Conclusions
	Acknowledgements
	References