Optimization of emulsification and the encapsulation process Stability of the primary emulsion

is necessary for successful stabilization of a multiple emulsion. Emulsifying agents lower the
surface tension of droplets and form a barrier to help prevent drop- let coalescence (Risch and
Reineccius 1988). Changes in the viscoelastic property of the ISP solution during MTGase
treatment are shown in Figure 3. Both the storage modulus G and stability of the emulsion
were increased with an increasing ISP concentration. A 10% ISP solution had a stable CIWE
with a high viscoelastic value. Both the increased viscosity and the protein concentration favor
the stability of the emulsion, in agreement with Hwang and others (1992). The concentration
of MTGase influenced the gel formation of ISP (data not shown). ISP was used as an
emulsifying agent for maintaining primary O/W emulsion (CIWE) stability. Other types of
emulsifying agents are also required for preparation of an O/W/O double emulsion.
Considering double emulsion stability and cap- sule productivity, Span 80 was the best
hydrophobic surfactant for microcapsule preparation. SDS-PAGE analysis was performed to
determine the effect of reaction time on the extent of MTGase polymerization of ISP (data not
shown). Molecular weights for native ISP were between 20 and 220 kDa. The intensity of
protein bands decreased with the reaction time up to 3 h and disappeared at 4 h, which
indicated ISP treated with MTGase resulted in formation of high molecular-weight poly- mer
aggregates, which did not enter the stacking gel. This was ac- companied by a decrease in band
intensities for the unpolymerized protein fractions. In contrast, no polymerization products
were observed for the control. These results indicate formation of inter- molecular cross-
linked polymers catalyzed by MTGase.
Physical properties of microcapsules Microcapsules had a narrow particle-size range (30 to 60
m) with a relatively uniform distribution (Figure 4). There were no sig- nificant differences (P >
0.05) in the mean diameters of MTGase- gelled, MTGase/heat-gelled, and heat-gelled
capsules. Smaller particles of approximately 2 m were present that were probably fragments
of denatured proteins. The morphology of the microcapsules containing fish oil was observed
under a scanning electron microscope (Figure 5). MT- Gase-gelled capsules and MTGase/heat-
gelled capsules had a spherical shape and exhibited a smooth, dent-free surface.
Microcapsules prepared from enzymatic gelation exhibited some sur- face pores. These pores
were probably the footprints of core drop- lets that were originally present at the surface and
were lost during the microencapsulation process (Lee and Rosenberg 2000a). Heat- gelled
capsules were roughly spherical in shape and had very po- rous and wrinkled surfaces (Figure
5c). According to Lee and Rosen- berg (2000a), these structural features suggest aggregation of
protein matrices rather than formation of a continuous film. Water-solubility of microcapsules
for controlled and sustained core release in an aqueous environment is of great importance to
the functionality of microcapsules. Microcapsule design requires limited or delayed water
solubility (Lee and Rosenberg 2000b). The water solubility of microcapsules is shown in Figure
6. MTGase- gelled capsules had remarkably lower water solubility. MTGase- gelled capsules
were not influenced by the incubation tempera- ture, whereas the solubility of heat-induced
capsules was influenced by the incubation temperature. The solubility of the capsules
increased with the temperature, influencing the micro- structural properties. The difference in
water solubility is probably related to differences in the cross-linking efficiency of protein-
based wall matrices obtained by enzyme-induced gelation and heat-induced gelation (Lee and
Rosenberg 2000a).
Controlled release and core retention The release mechanism of fish oil from microcapsules in
a pepsin solution was investigated because MTGase-gelled capsules were insoluble in the
water. The initial release of heat-gelled capsules was followed by 1st-order kinetics involving a
core material burst out from the capsules (Figure 7). MTGase-gelled capsules and
MTGase/heat-gelled capsules exhibited a sustained release of fish oil. These capsules showed
a half-order release profile up to 120 min of incubation time. The release rate shifted toward
1st-order kinetics as time progressed. The controlled-release mechanism of these capsules was
probably related to the surface characteristics of the microcapsules. The initial burst effect
of heat-gelled capsules can be interpreted as a fast release of fish oil from the surface pores of
the microcapsules (Cho 2000). At the end of the incubation time the fish-oil contents released
from the microcapsules were 78%, 81%, and 98% for MTGasegelled capsule, MTGase/heat-
gelled capsule, and heat-gelled cap- sule respectively, which was maintained over a 5-h
incubation time. These values indicated the total fish-oil retention for each microcapsule type.
Heat-induced gelation had the highest efficiency. In most cases, core retention for
microcapsules prepared by the double emulsion/subsequent gelation method is lower than for
spray- dried microcapsules because core losses during the process can occur during formation
of the O/W/O double emulsion during the cross-linking and washing stages (Lee and
Rosenberg 2000b). The low core retention in microcapsules prepared by enzyme-induced
gelation is probably because of the extended gelation time (4 h) compared with heat-induced
gelation and chemical cross-linking (20 to 40 min). Some fish oil may be flushed from wall
materials during the cross-linking stage. The high core retention of heat- gelled capsules may
be because of the shorter gelation time and the presence of effective emulsifier in the external
phase of the double emulsion, thus assuring the stability of the double emulsion

. Oxidative stability The p-anisidine reagent reacts with oxidation products, such as aldehydes
(principally 2-alkenals and 2, 4-dienals), producing a yellowish product. Hence, an increased p-
anisidine value (p-A.V.) indicates an increase in the amount of the oxidation product. The rate
of fat oxidation is a function of several factors, including water activity, availability of oxygen,
and the presence of antioxidants (Kim and Morr 1996). Figure 8 shows changes of the p-A.V.
value for nonencapsulated and encapsulated fish oil during storage. The increasing rate of the
p-A.V. value for nonencapsulated fish oil was much faster than for encapsulated fish oil. ISP
film acted as a good barrier, reducing the rate of fish-oil oxidation. Moreau and Rosen- berg
(1998) showed that the porosity of the matrix is affected by the fat core and encapsulant levels
used. However, oxidation of the fat core material did not occur over a 12-mo period with
protein wall matrices. Pearson correlation analysis was performed with the 3 variables of
mean particle size of microcapsules, the release rate of fish oil, and water solubility of the
microcapsules at 37 C at incubation temperatures (Table 1). The stability study by p-anisidine
produc- tion from encapsulated fish oil during storage was excluded from the variables for
Pearson correlation analysis because no significant differences were observed among the 3
encapsulated sam- ples. A strong correlation between the release rate of fish oil from
microcapsules and the water solubility of microcapsules incubat- ed at 37 C was detected.
Microcapsules having higher water solubility exhibited a faster release rate of fish oil. No linear
correlation was observed between the release rate or the water solubility and the mean
microcapsule particle size.
Conclusions MTGase modified intra- and inter-molecular interactions with protein-based wall
materials increasing the emulsion stability and rheological properties of proteins. ISP exhibited
the highest emulsion stability and gelling capacity. A CIWE (primary O/W emulsion) prepared
with 10% ISP was stable without any emulsifier.
Table 1Correlation analysis: Pearson correlation coefficients Particle Release Water size ( m)
rate (%) solubility (%) Particle size (m) 1.0000 0.3536 0.6166 Release rate (%) 0.3536 1.0000
0.9544 Water solubility (%) 0.6166 0.9544 1.0000
For gel formation of a double emulsion, a 4-h incubation time was required. There were no
significant differences in the mean diameter of microcapsules prepared with different gelation
methods. The size of capsules manufactured by the O/W/O double emulsification method was
not influenced by the gelation method. However, the capsule morphology was highly
influenced by the gelation meth- od. Microcapsules prepared by enzyme (MTGase)-induced
gelation were spherical in shape with a dent-free surface, while heat-gelled capsules were
wrinkled with a rough surface. MTGase-gelled capsules exhibited lower water solubility and
sustained release in a pepsin solution. However, the total oil retention for MTGase-gelled
capsules was lower than for heat-gelled capsules. Regardless of the gelation method,
encapsulated fish oil was more stable than non- encapsulated fish oil for each microcapsule
type, indicating that the encapsulation method presented herein is effective for protection of
the core material from oxidation. A microencapsulation process consisting of double
emulsification and subsequent gelation is suitable for protecting sensitive ingredients and for
controlled release in the food industry.