JFS: Food Engineering and Physical Properties

Water-Insoluble, Whey Protein–

based Microspheres Prepared
by an All–aqueous Process
M. ROSENBERG AND S.-J. LEE

ABSTRACT: Calcium-alginate coated microspheres consisting of whey proteins and a model sensitive core,
paprika oleoresin, were prepared using an all-aqueous process. Core-in-wall emulsions containing 20% or 25%
whey protein isolate and 15% to 50% (wt/wt) core were investigated. Retention of proteins and core during the
process ranged from 84.9% to 95.6% and from 91.4% to 95.7%, respectively. Results indicated that microspheres
were water-insoluble and the encapsulated sensitive core was effectively protected against oxidative deteriora-
tion. This protection could be attributed to the protein-based layer adsorbed at the oil/water interface. The matrix
of the microspheres exhibited microstructural features of an interactive composite-type material. Results sug-
gested the potential suitability of the microspheres as delivery systems for controlled core release in food appli-
cations.
Keywords: encapsulation, microspheres, oxidative stability, water solubility, whey proteins
Food Engineering and Physical Properties

Introduction of the protein-based devices exhibited desired functionality, they

D ifferent water-soluble microcapsules and microspheres con-

taining food ingredients and nutrients have been developed
(Jackson and Lee 1991; Arshady 1993; Shahidi and Han 1993; Gibbs
and others 1999; Augustin and others 2001). One of the main diffi-
culties associated with water-soluble delivery devices is their burst-
type core release characteristics. Many situations related to food
systems require a delivery device, in the form of microcapsule or
microsphere, from which the core can be released at a predeter-
mined rate at desired conditions at a desired time (Karel and
Langer 1988; Kirby 1991; Reineccius 1995). A prerequisite to ac-
complishing the latter is rendering the delivery devices water-insol-
uble (WIS). Although very common in pharmaceutical applications,
WIS delivery devices for controlled and/or sustained core release in
food applications have been developed only to a limited extent
(Karel and Langer 1988; Kirby 1991; Kumar 2000). Different natu-
ral biopolymers offer opportunities in developing the aforemen-
tioned desired delivery systems (Chasin and others 1990; Beaulieu
and others 2002).
The physicochemical, functional, and biodegradability proper-
ties of some proteins offer potential opportunities in developing
functional WIS delivery systems (Shah and others 1987; Raymond
and others 1990; Park and others 1998; Ugwoke and Kinget 1998).
Among these are albumins (Longo and others 1982; Chen and oth-
ers 1987; Gupta and Hung 1989; Arshady 1990; Lin and others 1993;
Park and others 1998), gelatin (Levy and Andry 1990; Oner and
Groves 1993), collagen (Rössler and others 1995), legumin (Irache
and others 1995), vicilin (Espeleta and others 1997), and bovine
caseins and albumins (Saleh and others 1989; Willmott and others
1992; Knepp and others 1993; Latha and Jayakrishnan 1994; Latha
and others 1994, 1995; Heelan and Corrigan 1997). Although many
MS 20030494 Submitted 8/31/03, Revised 10/2/03, Accepted 10/11/03. Au-
thors Rosenberg and Lee are with the Dept. of Food Science and Technology,
Univ. of California, Davis, CA 95616. Direct inquiries to author Rosenberg
(E-mail: mrosenberg@ucdavis.edu).
cannot be used in food applications. The preparation of these de-
livery systems commonly involves using organic solvents and/or
chemical cross-linking agents. This represents a difficulty when
food applications are considered, and thus a need to develop new
devices and technologies to meet this challenge exists.
Bovine whey proteins (WP) have been reported suitable wall
materials for preparing WIS microcapsules (Heelan and Corrigan
1998; Lee and Rosenberg 1999, 2000a, 2000b, 2000c, 2001; Satpathy
and Rosenberg 2003); however, the encapsulation processes in-
volved organic solvents and/or chemical cross-linking agents. To
overcome these difficulties, an “all-aqueous,” cross-linker-free
encapsulation processes with which WIS WP-based delivery devices
can be prepared had to be developed. It has been reported (Lee
and Rosenberg 2000a) that by highlighting the emulsification and
heat-gelation properties of WP, water-insoluble microcapsules
could be prepared in a process consisting of double emulsification
and subsequent heat treatment. However, an organic solvent had
to be used to remove the external oil-based phase from the outer
surfaces of the microspheres. To overcome this difficulty, the objec-
tive of our research was to investigate the formation and some
properties of WIS WP-based microspheres containing a model sen-
sitive core (paprika oleoresin [PO]), prepared by an all-aqueous
process.
Materials and Methods
Materials
Whey protein isolate (WPI) containing 95.6% protein was pur-
chased from Davisco Food Ingredients Int. (Le Sueur, Minn.,
U.S.A.). PO, containing natural extract of paprika dispersed in soy-
bean oil, was purchased from Kalsec (Kalamazoo, Mich., U.S.A.).
Alginic acid (sodium salt; low viscosity) was purchased from Sig-
ma Chemical Co. (St. Louis, Mo., U.S.A.). Anhydrous calcium chlo-
ride was purchased from Fisher Scientific Co. (Fair Lawn, N.J.,
U.S.A.).

FEP50 JOURNAL OF FOOD SCIENCE—Vol. 69, Nr. 1, 2004 © 2004 Institute of Food Technologists
Published on Web 1/23/2004 Further reproduction without permission is prohibited
Water-insoluble microspheres . . .
Emulsion preparation
WPI solutions (pH 6.8), containing 20% or 25% (wt/wt) WPI, were
prepared in deionized water containing 0.02% (wt/wt) sodium
azide. PO (at 20 °C) was emulsified into the WPI solutions at propor-
tions of 0%, 15%, 25%, 35%, and 50% (wt/wt). In all cases, the core-
in-wall emulsions (CIWE) were prepared at 20 °C according to the
2-stage emulsification and homogenization process that has been
reported by Lee and Rosenberg (2000a). The CIWE were denoted
20/0, 20/15, 20/25, 20/35, and 20/50; 25/0, 25/15, 25/25, 25/35,
and 25/50, respectively.
Preparation of microspheres
Calcium chloride (2 g) was added to a 100-g sample of CIWE,
and the mixture was stirred in the dark (25 °C) for 20 min. Ca-con-
taining CIWE (about 20 g) was dropped into a gently stirred dis-
persing medium consisting of 400 g of 1% sodium alginate (Na-Al)
solution to form a mixture of CIWE droplets dispersed in a Na-Al
solution. Stirring was carried out using a Nalgene floating magnetic
stirring bar (Fisher Scientific, Pittsburgh, Pa., U.S.A.). Dropping was
carried out using a 50-␮L pipette tip (Bio-Rad Laboratories, Her-
cules, Calif., U.S.A.) and a metering pump (Model P-1 LKB-Pump,
Pharmacia, Upsala, Sweden). Following the dropping stage, the
mixture was gently stirred for 5 min, and then the microspheres
were harvested by filtration (nr 4 filter paper, Fisher Scientific). The
recovered microspheres were washed 3 times with deionized water
and were then dispersed in 600 g of gently stirred 1% (wt/wt) CaCl2
solution at 85 °C. The mixture was stirred at this temperature for 20
min to induce gelation of the protein matrix of the microspheres.
The heat-treated microspheres were filtered and then rinsed 3
times with distilled water. Microspheres were kept dispersed in
deionized water containing 0.02% sodium azide pending analysis.
Throughout the preparation process, exposure to light was pre-
vented.
Analyses
Particle-size distribution. The particle-size distribution prop-
erties of CIWE was determined using a laser particle-size analyzer
(Malvern Mastersizer MS20, Malvern Instruments, Worcs, U.K.). A
2-mW He-Ne laser beam (633 nm) and a 45-mm focus lens were
used. The volume-size average d32 was used as the mean particle
size.
Composition analyses. For the determination of PO content of
microspheres, samples (0.5 g) were placed into 100-mL tubes,
ground with a glass rod, and then extracted in the dark with 40 mL
of acetone for 30 min. During the extraction, test tubes were shak-
en using a Model 7637 Roto-Troque Rotator (Cole-Parmer Instru-
ment Co., Chicago, Ill., U.S.A.). A preliminary study indicated that
the efficiency of the extraction method was higher than 99%. Fol-
lowing the extraction process, the supernatant was filtered through
a 0.45-␮m nylon membrane (Uniprep, Whatman Inc, Clifton, N.J.,
U.S.A.). The optical absorbance of the filtrate at 460 nm was deter-
mined using a Bausch and Lomb Spectronic 1001 spectrophotom-
eter (Spectronic Instuments Inc., Rochester, N.Y., U.S.A.), and the
amount of PO included in the extract was calculated using a stan-
dard curve of PO in acetone. Retention of PO during the micro-
spheres preparation process was expressed as the ratio (in %) of the
amount of PO included in unit mass of microspheres to the theo-
retical amount of PO in unit mass of microspheres, assuming 100%
retention. Total solids content and protein content of microspheres
was determined as previously reported by Lee and Rosenberg
(2000a). The proportion of calcium alginate (including all minerals)
in microspheres was calculated as the difference between total
solids content and the combined content of PO and proteins.
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Water solubility. Water solubility of both de novo and PO-con-
taining microspheres was investigated by monitoring the propor-
tion of soluble proteins released from the microspheres (Lee and
Rosenberg 2000a). Microcapsule samples (1 g) were placed in glass
vials containing 25 mL water at pH 7.0. To prevent microbiological-
related proteolysis, 0.02% sodium azide (Sigma Chemical Co.) was
added to the water. Capped vials were incubated at 25 °C, and the
concentration of soluble protein in the aqueous phase was deter-
mined after 1, 5, and 10 d of incubation. In all cases, a sample (0.75
mL) of the aqueous phase was filtered through UniPrep Syringeless
Filter Device (0.45 ␮m, Whatman Inc.) and protein content in the
filtrate was determined using the Bio-Rad D.C. Protein Assay kit
(BioRad, Hercules, Calif., U.S.A.). Bovine serum albumin (BSA) was
used as standard protein. Soluble protein was expressed as the
proportion (%, wt/wt) of total protein content included in the inves-
tigated microspheres.
Oxidative stability. The oxidative stability of PO encapsulated in
the microspheres was investigated, as a function of storage condi-
tions, by monitoring changes in the optical absorbance of the PO
content of microspheres (Beatus and others 1985). Samples (about
1 g) of microspheres prepared with CIWE 20/35 were placed in
capped glass vials containing 20 mL of 0.02% sodium azide solution
in deionized water. The dispersions were stored under light and in
the dark at 4 °C, 20 °C, and 30 °C. Periodically, vials were withdrawn,
microspheres were recovered from the dispersing medium, and
the optical absorbance of the PO extract, prepared as previously
Food Engineering and Physical Properties
described at standardized conditions, was determined spectro-
photometrically as previously described. Changes, per unit mass of
microspheres, in optical absorbance of the extract were expressed
as a percentage of the initial optical absorbance before the incuba-
tion. As a control, samples of unencapsulated PO (BPO) in the
amount equivalent to PO content of 1 g microspheres were stored
as previously described, and changes in their optical absorbance
were monitored as previously described, after dissolving the sam-
ples in acetone.
Structural features of capsules
The outer topography and inner structure of microspheres were
investigated by scanning electron microscopy (SEM). Microspheres
were fixed for 4 h in 4% glutaraldehyde in 0.1 M imidazole-phosphate
buffer at pH 7. The calcium alginate layer was dissolved during the
fixation process because of the chelating properties of the buffer.
The fixed microspheres were rinsed 3 times (15 min each) with 0.1 M
imidazole-phosphate buffer at pH 7, and then microspheres were
treated with 2% osmium tetroxide in 0.1 M imidazole-phosphate
buffer at pH 7 for 3 h. The fixed specimens were washed 3 times (15
min each) with the phosphate buffer and were then gradually dehy-
drated using a series of 35%, 50%, 70%, 80%, 95%, and 100% (vol/vol)
ethanol solutions. Dehydration was carried out for 20 min in each of
these solutions. The dehydrated microspheres were either treated
by critical point drying or freeze-fractured and then treated by critical
point drying, to allow studying of the outer topography and inner
structure, respectively. To study the structure of microspheres from
which the Ca-Al layer has not been removed, the microspheres were
treated as detailed previously, except that phosphate-containing
buffer was not used. In all cases, specimens were mounted onto a
SEM specimen stub using a 2-sided adhesive tape (Ted Pella, Red-
ding, Calif., U.S.A.). Specimens were coated with gold using a model
E-5050 Polaron Sputter Coater (Bio-Rad, San Jose, Calif., U.S.A.), and
then studied using an ISI DS-130 SEM (International Scientific In-
strument Inc., Pleasanton, Calif., U.S.A.) operated at 10 kV. Type 55
Polaroid film (Polaroid Corp., Cambridge, Calif., U.S.A.) was used to
produce micrographs.
Vol. 69, Nr. 1, 2004—JOURNAL OF FOOD SCIENCE FEP51
 Water-insoluble microspheres . . .
Statistical analysis
In all cases, experiments were carried in duplicate and analyses
in triplicate (n = 6). Significance of the results (P < 0.05) was deter-
mined by the ANOVA procedure included in the SigmaStat software
(Jandel Scientific software, San Rafael, Calif., U.S.A.).
Results and Discussion
Formation and structure of microspheres
The all-aqueous encapsulation process was designed to allow
preparing composite microspheres consisting of 3 major phases: (1)
a matrix, consisting of a whey proteins–based network, (2) a filler
phase consisting of PO droplets embedded in the network, and (3)
a surface layer of calcium alginate. The process involved 3 major
steps: (1) preparing a CIWE, (2) then using this emulsion, forming
“liquid core” microspheres, consisting of droplets of CIWE coated
with a skin of Ca-Al, and finally, (3) cross-linking the protein matrix
of the microspheres by heat treatment. The process thus exploited
both the emulsification and heat-induced gelation properties of
whey proteins.
Core retention, diffusion-driven core release, and the oxidative
stability of core that is susceptible to oxidation are all influenced by
the particle-size distribution properties of the CIWE (Moreau and
Rosenberg 1996, 1999; Lee and Rosenberg 2000a). Results depicted
in Figure 1 indicated that the particle-size distribution properties
of the CIWE were influenced (P < 0.05) by core load and to some
Food Engineering and Physical Properties
extent by WPI concentration. In most cases, CIWE exhibited unimo-
dal particle-size distribution. In some cases, such as with CIWE 20/
Figure 1—Particle-size distribution in core-in-wall-emul-
sions (CIWE), consisting of whey protein isolate (WPI) and
paprika oleoresin (PO)
FEP52 JOURNAL OF FOOD SCIENCE—Vol. 69, Nr. 1, 2004
50 and 25/50, a slight deviation from this distribution in the form
of a “shoulder” was evident. The presence of the shoulder indicat-
ed the presence of a subpopulation of core particles with a larger
mean particle diameter. In all cases, d32 < 1 ␮m was obtained; how-
ever, at a given WPI concentration, d32 in the CIWE increased (P <
0.05) with PO load included in the CIWE. Potentially, such an in-
crease could have been the result of decreasing the ration of pro-
tein-to-PO; however, in all cases, the amount of proteins was by far
larger than that needed to stabilize the core and thus did not rep-
resent a limiting factor (Wang 2004). The mean particle diameter in
CIWE 20/15, 20/25, and 25/15 was <0.5 ␮m and ranged from 0.28
to 0.47 ␮m, whereas that in all other CIWE ranged from 0.6 to 0.9
␮m.
The oxidative stability of core encapsulated in whey protein-
based wall matrices has been attributed to the formation of a WP-
based films at the oil/water (O/W ) interface (Moreau and Rosen-
berg 1996, 1999). The surface area of these interfacially adsorbed
films is determined by the specific surface area (SSA) of the CIWE.
Results indicated that at PO load of 15% and 25% (wt/wt), regard-
less of WPI concentration, SSA ranged from 10.12 to 21.25 m2/mL.
At a core load of 35% or 50% (wt/wt), the SSA was not affected by
WPI concentration (P > 0.05) and ranged from 6.55 to 7.34 m2/mL.
Particle-size distribution properties of CIWE, consisting of WPI and
melted anhydrous milk fat, prepared with CIWE compositions and
homogenization conditions similar to those investigated in the
present study, were not affected by core load (Young and others
1993; Lee and Rosenberg 2000a). The effect of core load observed
in the present study could be attributed to the high viscosity of the
core phase that adversely affected the homogenization efficiency.
In principle, the latter could have been improved by elevating the
homogenization temperature; however, in light of the sensitive
nature of PO, homogenization temperature was maintained at
20 °C.
The formation of stable microspheres was critically dependent
on rapid and effective formation of the Ca-Al “skin” around the dis-
persed CIWE droplets. The formation of such a skin could be ex-
pected to depend on rapid diffusion of Ca+2 ions, which were in-
cluded in the CIWE to the surface of the CIWE droplets to allow
rapid and effective formation of Ca-Al layer around these droplets.
The layer of Ca-Al had to stabilize the shape and size of the CIWE
droplets, pending solidification of the protein matrix during the
heat treatment. The Ca-Al layer had also to prevent significant pro-
tein loss to the dispersing medium and had to provide a mechan-
ical barrier capable of limiting core losses, pending the solidifica-
tion of the protein-based matrix. Additionally, the Ca-Al skin had to
prevent aggregation of the microspheres. The latter was likely to be
influenced by the stickiness of the skin surface.
Results of structure analysis (Figure 2) indicated that in all cases,
individual spherical or near spherical microspheres (ranging in di-
ameter from about 850 to about 1600 ␮m) were obtained. In all
cases, the wet Ca-Al skin did not lead to aggregation of the dis-
persed microspheres. Outer topography of microspheres was not
affected by the composition of the CIWE (Figure 2). The outer sur-
face of the microspheres was smooth and exhibited no cracks, sur-
face indentations, or visible pores, thus indicating the effective
formation of a continuous Ca-Al skin at the outer surfaces of the
dispersed CIWE droplets. Formation of teardrop-shaped micro-
spheres, which has been reported for liquid core microspheres con-
sisting of carbohydrate solution droplets coated with Ca-Al skin
(Nussinovitch and others 1996, 1997; Nussinovitch 2000), was not
observed in the present study. Results thus indicated that the com-
positions and process conditions used in the present study allowed
attainment of the desired spherical geometry of the microspheres.
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Water-insoluble microspheres . . .
The absence of surface indentations suggested that the micro-
spheres did not undergo uneven shrinkage that could potentially
be induced during the specimen-preparation process.
Results depicted in Figure 3a, 3c, and 3d indicated that, regard-
less of composition of CIWE, microspheres exhibited a “shell and
core” structure. In all cases, the outer surface of the microspheres
was coated with a continuous dense layer of Ca-Al, ranging in thick-
ness from about 45 to about 70 ␮m and formed a “shell” that coat-
ed the inner part of the microsphere. The inner part of the micro-
spheres, the matrix, consisted of the structures formed by the CIWE
constituents. The thickness of the Ca-Al layer was similar to that
reported by Nussinovitch and others (1996) for Ca-Al–coated car-
bohydrate-based spheres with liquid core. In some cases (Figure
3d), some small spherical voids were found in the matrix of micro-
spheres. These features could be attributed to footprint of some air
bubbles that have become entrapped in the CIWE.
Detailed inner structure of microspheres is depicted in Figure
3b, 3e, 3f, and in Figure 4. Regardless of core load, droplets of PO
were evenly distributed throughout the matrix formed by the whey
proteins. Results (Figure 3b) indicated that the matrix of de novo
microspheres exhibited the typical structural features of heat-in-
duced whey protein gel prepared at neutral pH (Langley and Green
1989a; Langton and Hermansson 1992; Foegeding and others
1995). Studying the inner structure of PO-containing microspheres
(Figure 3e and 4) revealed that in all cases, microspheres exhibited
structural features of lipid-containing WP-based composite gel
(Aguilera and Kessler 1989, Matsumura and others 1993; Mc-
Clements and others 1993; Chen and Dickinson 1999; Lee and
Rosenberg 2000a; Mor-Rosenberg and others 2003). These compos-
ites consisted of a continuous network formed by the aggregated
WP and a filler phase, which consisted of PO droplets embedded in
Figure 2—Outer topography of representative microspheres
prepared with core-in-wall emulsions (CIWE) 20/15 (a), 20/
35 (b), 25/15 (c), 25/35 (d), 25/50 (e), and 20/25 (f). Bar =
250 ␮m.
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this network. The size of core domains did not differ from that in the
CIWE and thus indicated that the preparation process, and espe-
cially the heat treatment did not adversely influence the original
core distribution properties of CIWE.
Results (Figure 4) indicated that the core droplets were coated
with films consisting of aggregated whey proteins (Figure 4c).
These interfacially adsorbed films were formed during the emulsi-
fication and homogenization stage of the process and exhibited
structural features similar to those of the interfacially adsorbed WP-
based films reported by Rosenberg and Lee (1993) for emulsions
consisting of WPI and milk fat and to those reported for composites
consisting of proteins and lipids ( Jost and others 1989; Yost and
Kinsella 1992; Lee and Rosenberg 2000a; Mor-Rosenberg and oth-
ers 2003). It must be noted that in most cases, the fracture plane
passed through most of the core domains. In such cases, the spec-
imen preparation process resulted in removal of the core droplets,
leaving behind only the parts of the interfacially adsorbed WP-
based films that were below the fracturing plane (Figure 4a, 4b, 4d,
4e, and 4f ). In a few cases, such as that presented in Figure 4c, the
fracture propagated through the microsphere in a way that left
some intact (or almost intact) protein-coated core domains, thus
revealing the structure of the protein-coated PO droplets, similar to
what has been reported by Rosenberg and Lee (1993) for chemically
fixed specimens consisting of WPI and milk fat.
Results (Figure 4) indicated that the protein layer adsorbed at
the surface of core droplets was physically connected to the WPI-
Food Engineering and Physical Properties
based continuous phase of the composite. This suggested that
proteins adsorbed at the surface of the PO droplets interacted with
Figure 3—Fractured microspheres prepared with core-in-
wall emulsions (CIWE) 20/0 (a), 20/35 (c), and 20/50 (d);
inner structure of microspheres prepared with CIWE 20/0
(b); structure details of microspheres prepared with CIWE
20/15 from which the Ca-Al layer has been removed (e and
f). CA = calcium-alginate layer; M = matrix; OS = outer
surface of the matrix. Arrows in D = voids. Bar = 250 ␮m
(a) 200 ␮m (c and d), and 5 ␮m (b, e, and f).
Vol. 69, Nr. 1, 2004—JOURNAL OF FOOD SCIENCE FEP53
 Water-insoluble microspheres . . .
the protein constituents included in the continuous phase of the
composite to form physical protein-based “bridges” (3D junctions),
similar to what has been suggested for composite gels consisting of
proteins and lipids (Aguilera and Kessler 1989; Yost and Kinsella
1992; McClements and others 1993; Chen and Dickinson 1999; Mor
and others 1999; Mor-Rosenberg and others 2003). The formation
of these junctions, formed by a combination of covalent (disulfide)
bonds and noncovalent interactions (such as hydrophobic and
hydrogen bonding), has been reported to mediate the anchoring of
the small lipid droplets in the protein network and to increase the
mechanical strength of composite (Jost and others 1986, 1989; Agu-
ilera and Kessler 1989; Langley and Green 1989b; Yost and Kinsel-
la 1992, 1993; McClements and others 1993; Hong and Dickinson
1996; Chen and Dickinson 1999; Mor and others 1999; Mor-Rosen-
berg and others 2003). Results thus suggested that the matrixes of
the PO-containing microspheres were of the interactive composite
type (Chen and Dickinson 1999; Mor and others 1999; Mor-Rosen-
berg and others 2003).
Although not investigated in the present study, it can be antic-
ipated that, in light of the above, the mechanical properties of the
microspheres would be affected, at a given WPI concentration, by
core load (Mor and others 1999; Mor-Rosenberg and others 2003).
In addition to “empty core domains,” results indicated the presence
of pores of different sizes and geometry shape, in the matrix of
microspheres (Figure 4). These pores reflected water-filled spaces
(pores) in the protein network of the matrix (Mor-Rosenberg and
Food Engineering and Physical Properties
others 2003). Results (Figure 3e and 3f ) indicated that the presence
of the Ca-Al layer at the surface of microspheres did not affect the
structural features of the outer surface of the protein-based matrix,
Figure 4—Inner structure of microspheres prepared with
CIWE 20/25 (a), 20/35 (b), 20/50 (c), 20/15 (d), 25/35 (e),
and 25/50 (f). M = matrix; OS = outer surface of the matrix
(see text). Black arrows = empty core domains revealing
parts of the interfacially adsorbed protein films. White ar-
rows = intact core droplets coated with protein layer. Bar
= 0.5 ␮m (c), 1 ␮m (a, b, and d), and 5 ␮m (e and f).
FEP54 JOURNAL OF FOOD SCIENCE—Vol. 69, Nr. 1, 2004
which was just below this layer. Studying the structural details of
the outer surface of the protein matrix, after the Ca-Al has been
removed (Figure 3f ), indicated that it consisted of strands of aggre-
gated WP and exhibited a typical structure of heat-induced WP-
based gel prepared at neutral pH (Langley and Green 1989a; Lang-
ton and Hermansson 1992; Foegeding and others 1995). Potentially,
creaming, or phase separation could have occurred in microspheres
before the solidification of matrix by the heat treatment. However,
results indicated that in all cases, core domains were evenly dis-
tributed throughout the protein matrix of microspheres and no
indications to suggest the latter could be found. These results thus
suggested that the viscosity and, probably, cohesive forces in the
CIWE were effective in preserving the original core droplets distri-
bution in the protein solution, pending the solidification of the
matrix. Results also indicated that core droplets did not undergo
flocculation that could have occurred in the presence of the includ-
ed calcium chloride. The latter could have reduce the negative
charge at the surface of the protein-coated core droplets to an ex-
tent where the magnitude of attractive interactions between these
droplets would be larger than that of the repulsive interactions.
As mentioned previously, the matrix of the microspheres con-
sisted of heat-induced WP-stabilized emulsion gel. The formation
of this gel represents the ultimate result of a cascade of physico-
chemical events, including protein unfolding and denaturation →
aggregation → gelation (Morr and Ha 1993; Boye and others 1997;
Verheul and Roefs 1998a, 1998b; Le Bon and others 1999; Mor and
others 1999; Mor-Rosenberg and others 2003). During heating, the
major whey proteins undergo unfolding, which also involves the
exposure and activation of the single thiol group of ␤-Lg. Heat treat-
ment, at conditions similar to those applied in our research (85 °C,
20 min, pH 6.8), has been reported to induce a rapid formation of
highly ordered stable structure stabilized by both covalent (disul-
fide) bonds and noncovalent protein-protein interactions, such as
hydrophobic and ionic interactions and hydrogen bonding (Mc-
Clements and others 1993; Iametti and others 1995; Verheul and
Roefs 1998a, 1998b). It has been reported that during heat-in-
duced gelation of whey proteins, the unfolded and activated WP
form molecular aggregates that interact with each other to form a
space-filling structure, a gel (Marangoni and others 2000). The
structural features exhibited by the matrix of the investigated mi-
crospheres were similar to those previously reported for whey pro-
tein-based microcapsules prepared by double emulsification and
subsequent heat gelation (Lee and Rosenberg 2000a).
Composition and components retention
Composition of microspheres is presented in Table 1, and the
retention of proteins and PO during the encapsulation process is
depicted in Figure 5. In all cases, protein retention higher than 84%
was accomplished. Protein retention in microspheres prepared with
CIWE containing 20% WPI (in the continuous phase) and 15% to
50% (wt/wt) PO was not affected by the core-to-wall ratio in CIWE
(P > 0.05) and ranged from 89.3% to about 95.0%. However, at WPI
content of 25%, protein retention was influenced by the core-to-wall
ratio (P < 0.05). At this WPI concentration, protein retention in mi-
crospheres prepared with 15% and 25% PO was higher (P < 0.05)
than that in microspheres prepared with 35% and 50% PO (Figure
5). Although not directly investigated in this study, these results
could be attributed to the inverse relationships between core con-
tent and heat transfer in the microspheres. It could be suggested
that the time needed to reach the temperature of gelation onset
increased with PO content. This, in turn, allowed a longer time dur-
ing which protein losses could occur. Potentially, diffusion-driven
protein losses could have occurred as long as the content of micro-
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Water-insoluble microspheres . . .

spheres was in the liquid state, that is, from the moment droplets Table 1—Composition of microspheres
of CIWE were dispersed in the sodium alginate solution pending Proportion Minerals + Al c
the stage where all of the protein constituents of the composite (%, wt/wt) CIWEa Protein POb PO/Protein d
become associated with the gel matrix. 20/15 47.1 38.4 14.5 0.82
It could be expected that protein losses would mainly occur be- 20/25 37.4 50.8 11.9 1.36
fore the formation of Ca-Al layer around the CIWE droplets and 20/35 30.5 58.8 10.7 1.93
then, to a lesser extent, during following stages of the process. Pro- 20/50 25.8 63.2 11.0 2.45
25/15 53.0 34.2 13.8 0.65
tein retention, during an encapsulation process in which heat-in-
25/25 42.3 45.2 11.5 1.07
duced gelation properties of proteins are exploited, is of critical 25/35 33.1 53.2 11.7 1.60
importance. In such a process, both the onset of gelation and the 25/50 26.9 62.2 10.9 2.31
formation of desired stable matrix structures are critically depen- a Core-in-wall emulsion, see text.
b Paprika oleoresin.
dent on maintaining protein concentration, inside the micro- c Total minerals and alginate content of microspheres.
spheres, at a level that is higher than the critical concentration for d Ratio of PO to proteins in microspheres.

gelation (Morr and Ha 1993; Le Bon and others 1999). Results of

composition analysis (Table 1) and the aforementioned results of
structure analysis suggested that in all cases, regardless of the com-
position of CIWE, this prerequisite had been met.
At the point where gel structure is formed, the gel point, only a
fraction of the proteins included in the system participate in form-
ing the aggregated 3D network. However, the ultimate spatial struc-
ture of the gel is determined at this stage ( Verheul and Roefs
1998a, 1998b; Marangoni and others 2000). At later stages of the
process, and even following the heating process, more of the aggre-
gated proteins interact via protein-protein interactions with the
existing primary structure and the ultimate gel structure and its
porosity is established (Verheul and Roefs 1998a, 1998b; Marango-
loss, pending, during, and probably following, heat treatment. It
could also be suggested that the presence of calcium ions in both
the CIWE and the dispersion medium during heat treatment also
facilitated protein retention. This could be attributed to the cold
gelation properties of denatured whey proteins (Beaulieu and oth-
ers 2002). It could thus be suggested that, potentially, part of the
proportion of the whey proteins that remained unassociated with
the gel matrix immediately following heat treatment, formed calci-
um-mediated “cold gel” domains, which stabilized this fraction of
the WP content of microspheres and limited its diffusion-related

Food Engineering and Physical Properties

ni and others 2000). It could thus be expected that, potentially,
some protein loss would occur following the heat treatment.
The overall high level of protein retention that was accomplished
with all the investigated systems suggested that the rapid forma-
tion of Ca-Al layers around the droplets of the CIWE established an
effective barrier that effectively limited diffusion-driven protein
Figure 5—Protein (white bars) and core (dark bars) reten-
tion in microspheres prepared with different core-in-wall
emulsions (CIWE). For a given WPI concentration, the level
of protein retention represented by bars marked by the
same lowercase letter is similar (P > 0.05). Similarly, for a
given WPI concentration, the level of core retention rep-
resented by bars marked by the same uppercase letter is
similar (P > 0.05).
mobility and hence potential loss. In light of this, it could be sug-
gested that protein losses mainly occurred before heat treatment
and probably before the formation of the Ca-Al layer around the
CIWE droplets.
Core (PO) retention (Figure 5) ranged from 91.4% to 95.9% and,
at a given WPI concentration in the CIWE, was not significantly af-
fected by the initial core load in the CIWE (P > 0.05). Core retention
in microspheres investigated in this study was similar to that re-
ported by Lee and Rosenberg (2000a) for WPI-based microspheres
prepared by double emulsification and subsequent heat gelation.
Potentially, core loss into the different dispersion media before
matrix solidification was governed by the combined influence of
the physicochemical properties of the CIWE, especially at stages
before the formation of Ca-Al films, by the rate at which Ca-Al films
were formed, and by the mechanical and barrier properties of these
films. The evident high level of core retention suggested that a rap-
id formation of Ca-Al film around the CIWS droplets dispersed in
the Na-Al limited core loss from the surface of the CIWE droplets.
At later stages of the process, these films continued to function as
a mechanical barrier that prevented core loss during the heat-in-
duced cross-linking processes.
In contrast to what has been observed in the present study, core,
anhydrous milk fat retention in WPI-based microspheres prepared
by double emulsification and heat gelation was significantly influ-
enced by the initial core load in the CIWE (Lee and Rosenberg
2000a). This difference could be explained in light of the functional
role of the Ca-Al layer around the microspheres investigated in the
present study in providing a physical barrier that limited the loss of
PO droplets from the surface of the microspheres into the dispers-
ing medium before matrix solidification. It could be expected that
increasing core load will increase the number of PO droplets at the
surface of the microspheres. In the absence of a barrier layer, such
as in the systems reported by Lee and Rosenberg (2000a), such
increase is likely to adversely affect core retention. However, the
insensitivity of core retention to initial core load in the present
study suggested the effectiveness of the Ca-Al films in preventing
such potential increase in core loss.

URLs and E-mail addresses are active links at www.ift.org Vol. 69, Nr. 1, 2004—JOURNAL OF FOOD SCIENCE FEP55
 Water-insoluble microspheres . . .
Results indicated no common trend relating particle-size distri-
bution in CIWE to core or protein retention. The parameter, core-to-
wall ratio, is important to the physical and functional properties of
microcapsules and microspheres. The core-to-wall mass ratio in
CIWE 20/15, 20/25, 20/35, and 20/50 was 0.75, 1.25, 1.75, and 2.50,
respectively, and that in microspheres prepared with these, CIWE
was 0.82, 1.36, 1.93, and 2.45, respectively. Similarly, the core-to-wall
ratio in CIWE 25/15, 25/25, 25/35, and 25/50 was 0.60, 1.00, 1.40,
and 2.00, and that in microspheres prepared with these CIWE was
0.65, 1.07, 1.6, and 2.31, respectively.
Results thus indicated that the high retention of both wall and
core constituents allowed the core-to-wall ratio in microspheres to
differ only slightly from that in the CIWE. These results also sug-
gested that the relative losses of core and wall constituents did not
introduce a significant deviation from the original core-to-wall ra-
tio. This information suggested that the investigated process allows
using the core-to-wall ratio in the CIWE to be used as a predictor to
the ultimate core-to-wall ratio in the microspheres. Such predica-
tion is of importance in light of the importance of core-to-wall ratio
in microspheres for controlled core release applications. Results
(Table 1) indicated that the proportion of solids other than core and
wall constituents in microspheres ranged from 10.9 to 14.5 (wt/wt,
dry basis).
Water solubility
The matrix of microspheres for controlled and/or sustain core
Food Engineering and Physical Properties
release in food applications has to remain water-insoluble pending
core release. Results (Figure 6) indicated that regardless of compo-
sition, both de novo and PO-containing microspheres were water-
insoluble. Monitoring the proportion of soluble proteins that was
released from microspheres over 10 d revealed that it ranged from
1.6% to 2.5% (wt/wt) of total protein content of microspheres (Fig-
ure 6). In most cases, changes with time in the proportion of soluble
proteins were small. In most cases the proportion of soluble proteins
released after 5 d was significantly larger than that released after 1
d (P < 0.05). In most cases, the proportion of soluble proteins ob-
Figure 6—Water solubility of microspheres
FEP56 JOURNAL OF FOOD SCIENCE—Vol. 69, Nr. 1, 2004
served after 10 d did not differ significantly from that obtained
after 5 d for a given WPI concentration. Although some differences
were observed between microspheres prepared with different
CIWE, no common trends relating composition of CIWE to water
solubility could be identified. Overall, the proportion of soluble
proteins released after 10 d from microspheres prepared with CIWE
containing 20% WPI was similar to that released from microspheres
prepared with the higher WPI concentration. This proportion ranged
from 1.8% to 2.5% (wt/wt) and from 1.6% to 2.25% (wt/wt), respec-
tively.
Results indicated that microspheres could be considered water-
insoluble and thus suitable, based on matrix properties, for con-
trolled and/or sustained core release in food applications. Overall,
the proportion of soluble proteins released from microspheres in-
vestigated in the present study was smaller than that reported by
Lee and Rosenberg (2000a) for WP-based microspheres prepared
by double emulsification and heat gelation. This difference prob-
ably reflected the contribution of some Ca-mediated gel, consist-
ing of heat-denatured WP, as explained previously. This, and the
majority of WP that was associated with the heat-induced gel, ren-
dered the matrix of the microspheres water-insoluble.
Oxidative stability
Results (Figure 7) indicated that the oxidative stability PO en-
capsulated in the investigated microsphere (EPO) was significantly
(P < 0.05) higher than that of bulk PO (BPO). The oxidative stabil-
ity of both EPO and BPO was inversely related to temperature and
storage time. Microspheres stored at 4 °C exhibited no oxidative
deterioration during 30 d, whereas oxidative deterioration of BPO
stored at 4 °C was evident after about 10 d of storage and reached
about 30% after 30 d. Results obtained at higher temperatures in-
dicated a very rapid and significant oxidative deterioration of BPO
and only a limited deterioration of EPO. Oxidative deterioration of
Figure 7—Oxidative stability of encapsulated (e) and bulk
(b) paprika oleoresin stored at 4 °C, 20 °C, and 30 °C under
light (see text for details). Microspheres were prepared
with CIWE 20/35.
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Water-insoluble microspheres . . .
BPO stored at 20 and 30 °C was detected after less than 5 d and
progressed at a very fast rate. Results obtained with BPO stored at
20 °C and 30 °C indicated oxidative deterioration of 50% after about
9 d and 13 d, respectively, and of about 90% and 97% after 30 d,
respectively. Results obtained with EPO stored at these tempera-
tures indicated a very slow and limited deterioration that reached
about 11% and 15% after 30 d at 20 and 30 °C, respectively.
The carotenoids constituents of PO are highly susceptible to
oxidation that is promoted by temperature and light (Beatus and
others 1985). Results obtained with EPO indicated that the encap-
sulation of the PO in the WP-based microspheres provided this
sensitive core with effective protection against oxidative deteriora-
tion. This protection could be attributed to the reported function-
ality of WP-based films adsorbed at the O/W interface of the lipid
core in protecting the core against oxidation (Moreau and Rosen-
berg 1996, 1999; Wang 2004). The role of WP-based films adsorbed
at O/W interface in protecting lipids against oxidation has been
reported for both heat-treated and untreated WP-stabilized emul-
sions (Moreau and Rosenberg 1996; Wang 2004). Heat treatment
conditions similar to those used in the present study have been
reported to induce formation of well developed, very stable WP-
based films, stabilized by both disulfide and noncovalent bonds,
at the O/W interface of WP/lipids emulsions (Wang 2004).
The formation and stabilization of the interfacially adsorbed
films, similar to those depicted in Figure 4, has been reported to
involve adsorption and natural cross-linking of WP during both
emulsification/homogenization and subsequent heat treatment
(Wang 2004). It has been indicated that at surface excess higher
than the monolayer level, these films provided lipids with excellent
protection against oxidation (Wang 2004). Results pertinent to the
oxidative stability of PO and results of structure analysis of micro-
spheres (Figure 4) thus agree with those reported by Moreau and
Rosenberg (1996, 1999) and by Wang (2004). In light of role of the
WP-based interfacially adsorbed films in governing the oxidative
stability of the sensitive core, and when the relationships between
protein surface excess and mean particle is considered, the advan-
tage of reducing the size of the core droplets in the CIWE as much
as possible is clear. Maintaining the mean particle size as small as
possible provides means to establish the largest SSA available for
protein adsorption possible. The latter can be expected to enhance
the oxidative stability of a given volume of encapsulated core.
Conclusions
A n all-aqueous process allowed preparing of Ca-alginate coated,
WIS microspheres consisting of whey proteins and a model sen-
sitive core, PO. The investigated process provided means to attain
high retention of both matrix constituents, proteins, and of core.
The microstructural features of the investigated microspheres in-
dicated the interactive composite nature of the microspheres ma-
trix. Results indicated the effectiveness of the Ca-Al layer, at the
surface of the microspheres, in maintaining the shape and size of
the CIWE droplets pending the solidification of the matrix. Results
also suggested the functionality of this layer in governing core and
protein retention during the process. The sensitive core encapsu-
lated in the microspheres was effectively protected against oxida-
tive deterioration, even at conditions that are known to promote
oxidation. The functionality of the microspheres in providing this
protection could be attributed to the formation of whey protein-
based films at the O/W interfaces. The investigated process and the
resulting functional microspheres provide means to overcome dif-
ficulties pertinent to preparation of water-insoluble, protein-based
microspheres for food applications. The use of microspheres, sim-
ilar to those reported here, has to be evaluated in light of their po-
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tential impact on the textural and sensorial properties of the prod-
uct into which they are incorporated. For some applications, such as
cereal bars, and so forth, the incorporation of microspheres with a
0.5- to 1-mm dia does not present a difficulty. However, in other
applications, microspheres smaller than those reported here are
needed. It has also to be noted that the encapsulation process re-
ported in this study involves a heat treatment at a relatively high
temperature. It is thus clear that the process may not be suitable for
encapsulation of core material that is very heat labile.
Results indicated that the matrix of the microspheres was water-
insoluble and thus suggested the potential suitability of the micro-
spheres to serve as a delivery system for controlled and/or sus-
tained core release in food applications. Although not directly
investigated in this study, data indicating the biodegradability of
heat-treated whey protein-based matrices have been reported and
thus suggested the degradability of the microspheres reported
here in the gastrointestinal tract. Results of the study have further
highlighted the suitability of whey proteins to serve as functional
encapsulating agents.
Acknowledgments
The research was funded by a grant provided by The Center for
Advanced Processing and Packaging Studies (CAPPS, NSF).
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