Food Chemistry 180 (2015) 150–155

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Kinetics of immobilisation and release of tryptophan, riboflavin and

peptides from whey protein microbeads
Graham J. O’Neill, Thelma Egan, Jean Christophe Jacquier, Michael O’Sullivan, E. Dolores O’Riordan ⇑
Food for Health Ireland, UCD Institute of Food and Health, University College Dublin, Belfield, Dublin 4, Ireland

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

Article history: This study investigated the kinetics of immobilisation and release of riboflavin, amino acids and peptides
Received 26 May 2014 from whey microbeads. Blank whey microbeads were placed in solutions of the compounds. As the vol-
Received in revised form 10 January 2015 ume of microbeads added to the solution was increased, the uptake of the compounds increased, to a
Accepted 31 January 2015
maximum of 95% for the pentapeptide and 56%, 57% and 45% for the dipeptide, riboflavin and tryptophan
Available online 7 February 2015
respectively, however, the rate of uptake remained constant. The rate of uptake increased with increasing
molecule hydrophobicity. The opposite was observed in the release studies, the more hydrophobic
Keywords:
compounds had lower release rate constants (kr). When whey microbeads are used as sorbents, they
Encapsulation
Release
show excellent potential to immobilise small hydrophobic molecules and minimise subsequent diffusion,
Kinetics even in high moisture environments.
Peptide Ó 2015 Elsevier Ltd. All rights reserved.
Hydrophobic

1. Introduction
The development of functional foods can be made challenging
due to the sensitivity of bioactive compounds to a range of food
processing and storage conditions including: acidic pH, activity of
enzymes, exposure to oxygen and light as well as the poor sensory
appeal of some bioactives (Chen & Subirade, 2006). Encapsulation
has been shown to be a successful technique in protecting a range
of bioactives against these conditions (de Vos, Faas, Spasojevic &
Sikkema, 2010). Encapsulation techniques that have been used to
achieve this include emulsification, coacervation, spray drying
and entrapment in hydrogels using a range of encapsulating mate-
rials. The materials (Chitosan, Alginate, Pectins and Soybean oil)
can often be used in a pure form or mixed with other polymers
depending on where in the gastrointestinal tract the bioactive is
to be released (de Vos et al., 2010).
Whey protein hydrogels have been used successfully to encap-
sulate a range of potential functional ingredients including
riboflavin (Egan, Jacquier, Rosenberg, & Rosenberg, 2013a), caffeine
(Gunasekaran, Ko, & Xiao, 2007), probiotics (Doherty et al., 2012)
and anthocyanins (Betz & Kulozik, 2011). In addition the cold set
gelation technique has been shown to be an effective technique
for the encapsulation of a range of compounds and is also ideal
⇑ Corresponding author. Tel.: +353 1 716 2847.
E-mail address: dolores.oriordan@ucd.ie (E. Dolores O’Riordan).
for heat sensitive bioactives (Chen & Subirade, 2006; Déat-Lainé,
Hoffart, Garrait, & Beyssac, 2013).
However, studies using this technique often add the bioactive to
the microbead forming solution prior to calcium induced gelation
which can result in diffusional losses of the bioactive during the
crosslinking and washing steps. Addition of compounds to the
microbead forming solution can also result in gelling of the bead
forming solution (Wells & Sheardown, 2007).
To overcome this, preformed whey microbeads were examined
as sorbents for a range of bioactive compounds. The use of
microbeads as sorbents has been studied with polymers such as
alginate and dextran (Chan & Neufeld, 2010; Chan, Yim, Phan,
Mansa, & Ravindra, 2010; Schillemans, Verheyen, Barendregt,
Hennink, & Van Nostrum, 2011), but limited studies using whey
protein microbeads as sorbents have been conducted (Déat-Lainé
et al., 2013). The sorption method has many advantages including
no diffusional loss of the bioactive during crosslinking and the
gentle conditions used which avoid high heat and shear. This tech-
nique can also facilitate large scale synthesis of blank microbeads
and subsequent loading with an array of different food bioactives
for a range of applications (Wells & Sheardown, 2007).
Déat-Lainé and co-workers (2013) reported high levels of
encapsulation by placing lyophilised WPI-alginate microparticles
in solutions of insulin. O’Neill et al. (2014) expanded on this
sorption approach by encapsulating a range of compounds in pre-
formed whey microbeads. This research showed that hydrophobic
interactions between the microbeads and the compounds to be

http://dx.doi.org/10.1016/j.foodchem.2015.01.131
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
 G.J. O’Neill et al. / Food Chemistry 180 (2015) 150–155
encapsulated could be used to increase the total percentage encap-
sulation. In addition, it was shown that the entrapment of the
compounds within the microbeads was occurring via a process of
partition. Egan, O’Riordan, O’Sullivan, and Jacquier (2014) encapsu-
lated charged peptides in preformed whey protein microbeads
using a similar sorbent method. Adjusting the pH of peptide solu-
tions to facilitate an electrostatic interaction between the peptides
and the microbead resulted in high levels of peptide encapsulation.
However, the studies to date using the sorption approach did
not address the kinetics of encapsulation. To successfully use
preformed microbeads for encapsulation, an understanding of the
encapsulation kinetics is required. This would allow the mechan-
ism of the encapsulation process to be identified as well as the
determination of the rate at which encapsulation occurs. Factors
that influence the rate constant could also be identified and poten-
tially altered to increase the rate of encapsulation (Meites, 1981).
Additionally, the point at which the encapsulation process reaches
equilibrium could be identified, thereby improving the efficiency of
the process. An understanding of the kinetics of bioactive release
from the microbeads is equally important as it predicts when,
where and how a bioactive will be released. Early release of a
bioactive in either a food product or in the stomach would result
in the bioactive receiving no protection against digestion.
The purpose of this study was to examine the kinetics of bioac-
tive encapsulation in whey microbeads in order to determine if
using preformed microbeads is an efficient method of encapsula-
tion. In addition, factors that influence the rate of encapsulation
including volume ratio (the ratio of microbead to bioactive solu-
tion) and hydrophobic interactions were examined. The release
kinetics of the bioactives form the beads were also studied in order
to determine if whey microbeads would be suitable for incorpora-
tion into a food matrix.
2. Materials and methods
2.1. Materials
Methanol-HPLC grade, tryptophan, riboflavin, monosodium
phosphate, disodium phosphate and sodium azide were purchased
from Sigma Aldrich (Ireland). The peptides Phe-Trp (Mw 351) and
Leu-Trp-Met-Arg-Phe (Mw 751) were purchased from Bachem
(Bubendorf, Switzerland). Demineralised whey protein isolate
(WPI) containing 2% ash and 92% (dry weight) protein as deter-
mined by the Macro-Kjeldahl method (AOAC, 1997) was purchased
from Davisco Food Ingredients Int. (Le Sueur, MN, U.S.A.). Calcium
chloride dihydrate was purchased from Merck (Darmstadt, F.R.,
Germany).
2.2. Manufacture of blank WPI microbeads
The cold set WPI microbeads were manufactured as described
by Egan, Jacquier, Rosenberg, and Rosenberg (2013b). The WPI
was rehydrated in deionised water (containing 0.02% sodium
azide) under magnetic stirring to 10% w/w on a protein basis,
and left to stand overnight at 4 °C to allow hydration of the pro-
teins. The solution was denatured by heating to 80 °C and holding
for 30 min in a shaking water bath (model DMS360, Fisher Scien-
tific, Dublin, Ireland) and subsequently cooled to 25 °C in ice water.
The heat denatured WPI (pH 6.8, 15 ml) was dispensed at 20 °C
through a 10 ll pipette tip using a variable speed transfer pump
(Gilson minipuls 2, Villiers-le-Bel, France) at a rate of 0.6 ml per
minute into 100 ml of 100 mM calcium chloride cross-linking solu-
tion to produce microbeads. Microbeads were 1.8 mm in diameter
with a pI of 4.7. These beads were stored in the calcium chloride for
24 h at 4 °C followed by multiple rinses with deionised water.
151
2.3. Encapsulation kinetics
Microbeads (2, 3, 4 or 6 ml) were placed in an Erlenmeyer flask
and volumes of 10 or 15 ml of 0.2 g/L solutions of tryptophan,
dipeptide or pentapeptide or 0.02 g/L riboflavin, were pipetted into
the flask. The volumes were varied to examine the effect of the
volume ratio (the ratio of microbead to bioactive solution) on
encapsulation rate (ke). The solutions were stirred at low speed
via magnetic stirring bars to ensure even distribution of the com-
pounds. Samples (100 ll) of the solution were taken at 0, 1, 2, 3,
5, 7, 10, 13, 15, 20, 30, 40, 50, 60, 70 and 80 min, and analysed
by HPLC as per Section 2.6 to determine the concentrations of
compounds in solution. The entrapment of bioactives in the
microbeads involves a process of immobilisation. The terms encap-
sulation will be used interchangeably with the term immobilisa-
tion to describe the uptake of bioactives into the microbeads. The
% of bioactive immobilised will be expressed as the E (%), calculat-
ed using Eq. (1):
M 0  M aq
E ð%Þ ¼  100 ð1Þ
M0
where Maq is mass of solute in solution at time t, and M0 is the mass
of solute in the initial solution.
The encapsulation kinetics of tryptophan, riboflavin, dipeptide
and pentapeptide were fitted to the first order kinetic model adapt-
ed from Reis, Guilherme, Rubira, and Muniz (2007). The kinetic val-
ues for the first order model were calculated using Eq. (2):
 . !
V aq
ke V t Emax
E ð%Þ ¼ Emax 1  e bead
ð2Þ
where Emax is the maximum bioactive decrease in concentration in
the surrounding solution at equilibrium Co  Ceq, ke is the encapsu-
lation rate constant (min1), Vaq is the volume of bioactive solution,
Vbead is the volume of microbeads and t is time.
2.4. Release kinetics
Initially microbeads (4 ml) where placed in solutions of either
tryptophan, riboflavin, dipeptide or pentapeptide at a volume ratio
of 0.2 for 24 h under magnetic stirring to load the microbeads. The
volume ratio was defined as the volume of microbeads (Vbead)
divided by the volume of solution (Vaq). Concentrations of 0.5 g/L
were used for tryptophan, dipeptide and pentapeptide, and
0.05 g/L were used for the riboflavin solution. Concentration of
model bioactive in the initial solution and after loading were anal-
ysed by HPLC to determine the mass of compound in the
microbead after loading. Release from microbeads was conducted
by adding 2, 3, 4 or 6 ml of loaded microbeads to 10 ml of deionised
water under stirring, and taking samples (100 ll) at appropriate
time points. All samples were analysed by HPLC for molecule con-
centration as described in Section 2.6. The release of the model
bioactive was described according to Reis et al. (2007) using Eq.
(3):
 
Release ð%Þ ¼ Rmax 1  eðkr t=Rmax Þ ð3Þ
where Rmax is the maximum bioactive in solution at equilibrium, kr
is the release rate constant (min1) and t is time.
2.5. Application of partition theory to the release of compounds from
the microbeads
Partitioning refers to the distribution of a compound between
two immiscible phases. In the experiments presented the whey
microbead and the release medium are the two phases. At equilib-
152 G.J. O’Neill et al. / Food Chemistry 180 (2015) 150–155
rium the distribution of the compounds between both phases can
be expressed as
K 50
½Aaq ¡ ½Abead
Where [A]aq and [A]bead is the solute concentration in the aque-
ous phase and microbead phase respectively. Partition theory can
relate the affinity of the encapsulated compound to either the
whey microbead or the release medium. The equilibrium constant
(K) can be calculated using Eq. (5).
½A M bead V aq
K ¼ bead ¼  ð5Þ
½Aaq M aq V bead
where [A]bead and [A]aq is the solute concentration in the microbead
and the aqueous phase respectively, and Mbead and Maq is the mass
of solute in the microbead and aqueous phase, respectively. Vbead
and Vaq is the volume of the microbead and aqueous solution
respectively. K is obtained from the slope of the plot of Mbead/Maq
against Vbead/Vaq.
2.6. HPLC analysis
All samples were analysed on an Aglient 1200 series HPLC. An
Agilent Eclipse XDB-C18 (150 mm  4.6 mm i.d.; 5 lm particle
size) column with a C18 guard column (Phenomenex, Macclesfield,
UK) was used for all analysis. Detection was conducted at 210 nm
for tryptophan and the peptides, and at 270 nm for riboflavin. A
sample injection volume of 10 ll was used for tryptophan and pep-
tides, and 20 ll for riboflavin, amber vials were used to prevent
photo degradation. All analysis was conducted using a mobile
phase of methanol and a 25 mM phosphate buffer; pH 6 for
riboflavin, and pH 7 for amino acids and peptides. The ratio of
methanol to phosphate buffer used for analysis were 7:93 for tryp-
tophan, 40:60 for riboflavin and 50:50 for the dipeptide and pen-
tapeptide. To determine a value of molecule hydrophobicity the
capacity factor (k0 ) for each of the compounds was calculated using
HPLC. The capacity factor was calculated using a mobile phase of
50:50 methanol:phosphate buffer 25 mM (pH 7). The capacity fac-
tor was calculated from Eq. (6) (Serban & Victor, 2013).
0 tr  t0
k ¼
t0
where tr is the retention time of the compound and t0 is the void
volume of the solvent peak.
2.7. Statistical analysis
All experiments were conducted in triplicate. The data points
shown are experimental and the models are calculated by averag-
ing the constants from each of the replicates. Statistical analysis
was conducted with SAS software (Systat Software, SanJose, CA,
U.S.A.) using Tukey’s pair wise comparison to determine significant
differences between treatments. Values with the same letter are
not significantly different at p 6 0.05.
3. Results and discussion
3.1. Encapsulation kinetics of tryptophan, riboflavin and peptides in
blank microbeads
The kinetics of tryptophan encapsulation (E (%)) in blank
microbeads at a range of volume ratios is shown in Fig. 1. The vol-
ume ratio is the ratio of the volume of microbeads submerged in a
volume of tryptophan solution. As volume ratio increases, the
quantity of protein relative to amount of tryptophan solution
increases. Tryptophan uptake into the microbeads was rapid,
60
ð4Þ
Encapsulation (%)
40
30
20
10
0
0 10 20 30 40
Time (mins)
Fig. 1. The percentage encapsulation of tryptophan into whey microbeads as a
function of time at volume ratios (Vbead/Vaq) of 0.13 (d), 0.2 (s), 0.3 (j), 0.4 (h), and
0.6 (N). The line represents model fits to first order kinetics.
reaching an equilibrium concentration in the microbeads after
approximately 20 min.
Volume ratio did influence the encapsulation maximum (Emax)
of tryptophan, at equilibrium an Emax of 16 ± 1.1% was obtained
at a volume ratio of 0.13 while at a volume ratio of 0.6 the Emax
was significantly (p 6 0.05) higher at 45 ± 1%. The Emax for trypto-
phan in microbeads increased with increasing volume ratio and
was significantly different at each volume ratio (Table 1).
To evaluate the uptake of tryptophan into blank microbeads the
first order model adapted from Reis et al. (2007) was fitted to the
data. At all volume ratios the model showed an excellent fit
(R2 > 0.97) to the experimental data (Fig. 1). No significant differ-
ence (p 6 0.05) was noted between the ke values at different vol-
ume ratios indicating volume ratio did not influence the rate of
tryptophan uptake into the microbeads. An average rate constant
(ke) of 0.29 min1 was obtained for tryptophan uptake into the
microbeads. Therefore, by increasing the volume of microbeads
in tryptophan solution a greater Emax was obtained. However, the
rate of tryptophan uptake occurs independent of volume ratio.
ð6Þ The effect of volume ratio on the encapsulation of riboflavin, a
dipeptide and a pentapeptide was examined and the results are
summarised in Table 1. For each of the compounds the Emax at
equilibrium increased with increasing volume ratio, Emax values
of 57 ± 2.6, 56 ± 2.8 and 95 ± 2.4 were obtained respectively, at a
volume ratio of 0.6. Similarly to the results observed with trypto-
phan, the rate constant for uptake of each of the compounds was
constant and independent of volume ratio. The average rate con-
stants ke obtained for riboflavin, dipeptide and the pentapeptide
were 0.4, 0.38 and 0.69 min1, respectively.
The kinetics of riboflavin, dipeptide and pentapeptide encapsu-
lation at a volume ratio of 0.4 were determined. Uptake of each of
the compounds was rapid with loading occurring in less than
40 min. The encapsulation of the three compounds fitted first order
kinetics, with the pentapeptide data showing an excellent fit to the
model. The initial stages of riboflavin and dipeptide uptake were
faster than that expected from a first order profile, however the
good R2 values of 0.94 and 0.95 respectively, suggested uptake of
both is a first order kinetic process.
It is evident from Table 1 that when comparisons are made
between the compounds, differences in the E (%) and rate constant
ke at the same volume ratio exist. At all volume ratios, tryptophan,
on average had the lowest E (%) values and the slowest rate
constant (ke) values while the pentapeptide had the highest.
The hydrophobicity of the molecules was calculated by measur-
ing their capacity factor (k0 ). The k0 values of tryptophan, riboflavin,
 G.J. O’Neill et al. / Food Chemistry 180 (2015) 150–155 153

Table 1
Encapsulation rate constants (ke) and encapsulation maximum (Emax) values for tryptophan, riboflavin, dipeptide and pentapeptide in whey protein microbeads.

Volume ratio Tryptophan Ribofavin Dipeptide Pentapeptide

1 1 1
Emax (%) ke (min ) Emax (%) ke (min ) Emax (%) ke (min ) Emax (%) ke (min1)
a a a a a a a
0.13 16.3 ± 1.1 0.30 ± 0.09 24.5 ± 0.7 0.45 ± 0.09 23.6 ± 3.7 0.44 ± 0.14 87.5 ± 0.7 0.72 ± 0.04a
0.2 19.6 ± 2.3b 0.26 ± 0.16a 33.3 ± 0.5b 0.40 ± 0.01a 30.6 ± 3.0b 0.35 ± 0.09a 91.3 ± 0.2b 0.72 ± 0.04a
0.3 29.3 ± 0.5c 0.29 ± 0.06a 41.6 ± 1.5c 0.46 ± 0.13a 39.3 ± 2.0c 0.37 ± 0.03a 92.5 ± 0.6bc 0.70 ± 0.06a
0.4 36.3 ± 1.5d 0.31 ± 0.04a 50.3 ± 0.5d 0.34 ± 0.06a 46.1 ± 0.1d 0.35 ± 0.03a 93.5 ± 0.7cd 0.67 ± 0.05a
0.6 45.0 ± 1.0e 0.32 ± 0.03a 57.0 ± 2.6e 0.37 ± 0.05a 56.3 ± 2.8e 0.39 ± 0.16a 95.1 ± 2.4d 0.67 ± 0.06a

Mean values ± standard deviation denoted by the same letters within a column are not significantly different at p P 0.05.

dipeptide and pentapeptide were 0.74, 1.8, 2.58 and 21.47, respec-
tively, reflecting an increase in hydrophobicity. The corresponding
increase in average rate constant (ke) values (0.29, 0.4, 0.38 and
0.69) for these compounds (tryptophan, riboflavin, dipeptide and
pentapeptide), suggests that hydrophobic interactions influence
the rate at which uptake into the microbeads occurs.
The migration of amino acids or peptides from solution into
microbeads has previously been shown to be a partition process
where the extent of the compounds migration is influenced by
the affinity of the compound for the whey microbead. The more
hydrophobic the amino acid or peptide the greater the mass of
compound that resided within the microbead phase at equilibrium
(O’Neill et al., 2014). The data in Table 1 expands on this by show-
ing that the rate of migration is also influenced by this hydrophobic
interaction, the stronger the affinity the faster the rate of uptake.
As the whey protein used in the microbeads was denatured, the
hydrophobic regions in proteins such as b-lactoglobulin, which are
usually located within the folded region of the protein become
exposed. As a result, hydrophobic domains may be formed within
the microbead which in turn facilitate migration of hydrophobic
compounds from the external solution into the microbead. b-lac-
toglobulin which is a lipocalin, can facilitate small hydrophobic
ligands and it has been shown to encompass a range of small mole-
cules including retinol, vitamin D2 and cholesterol (Kontopidis,
Holt, & Sawyer, 2004).
Studies using blank whey based microparticles have shown that
adsorption/absorption of compounds into such ‘blank’ microparti-
cles can be a more effective method for encapsulating compounds
such as insulin and riboflavin than adding the compounds to the
microparticle forming solution (Déat-Lainé et al., 2013; O’Neill
et al., 2014). From the kinetics studies on tryptophan, riboflavin
and peptides presented in this paper it may be concluded that
loading of the microbeads occurs over a short period of time,
increasing the potential of blank microbeads as a viable method
of encapsulating small hydrophobic molecules.
reported to follow a first order release profile (Gunasekaran
et al., 2007).
Tryptophan release occurred in a short period of time reaching
release equilibrium in less than 30 min. Release of the other com-
pounds was slower than that of tryptophan, but was none the less
still quite rapid with both riboflavin and the dipeptide reaching
release equilibrium within one hour with 48% and 56% of the
encapsulated species released respectively. Release of the pen-
tapeptide was considerably slower than all of the other compounds
as it only reached release equilibrium after six hours. There was an
initial release of 2.4% in the first 15 min followed by a subsequent
release of a further 1.6% before reaching equilibrium after 6 h. The
rate constants (kr) for tryptophan, riboflavin, dipeptide and pen-
tapeptide release are shown in Table 2. The volume ratio did not
have a significant effect (p P 0.05) on the release rate constant
for any of the compounds. Average rate constants (kr) of 0.22,
0.09, 0.1 and 0.014 min1 were obtained for tryptophan, riboflavin,
dipeptide and pentapeptide respectively.
Previous studies have shown that hydrophobic interactions
between compounds and whey microbeads can influence the E
(%) at equilibrium, with E (%) increasing with increasing molecule
hydrophobicity (O’Neill et al., 2014). Interestingly the same
hydrophobic interactions appear to influence both the rate con-
stant for encapsulation (ke) and release (kr). The rate of uptake
increased with increasing molecule hydrophobicity whereas the
rate of release decreased with increasing molecule hydrophobicity
(Table 2). These results suggest that hydrophobic interactions are
important as they increase both the Emax and the rate of uptake
while the stronger the interaction with the microbead the slower
the rate of release.
70
60

3.2. Release kinetics of tryptophan, riboflavin and peptides from

50
microbeads
Release %

An understanding of the release kinetics of a molecule from 40

an encapsulation system is important in order to establish if it

is suitable for its intended use. Hydrophobic interactions have 30
been reported as being able to substantially slow the release
profile of a drug (Pavli, Baumgartner, Kos, & Kogej, 2011). There- 20
fore, the effect of molecule hydrophobicity on release rate from
whey protein microbeads was examined by initially encapsulat- 10
ing tryptophan, riboflavin, a dipeptide and pentapeptide and
subsequently studying the release kinetics. The release kinetics 0
of all compounds showed an excellent fit to a pseudo first-order 0 10 20 30 40 50 60 70

kinetic model. As volume ratio was not observed to influence the Time (mins)
time taken to reach equilibrium the release kinetics are shown Fig. 2. Release of encapsulated tryptophan (d), riboflavin (N), dipeptide (s) and
for the compounds at just one volume ratio (0.4) (Fig. 2). The pentapeptide (4) from whey microbeads at a volume ratio (Vbead/Vaq) of 0.4. The
release of caffeine from whey protein gels has previously been solid line represents the first order model fit.
154 G.J. O’Neill et al. / Food Chemistry 180 (2015) 150–155

Table 2
Release rate constants for tryptophan, riboflavin and peptides from whey microbeads.
Volume ratio Tryptophan Riboflavin Dipeptide
kr (min1) kr (min1) kr (min1)
0.13 0.22 ± 0.04a 0.09 ± 0.008a 0.09 ± 0.002a
0.2 0.21 ± 0.03a 0.09 ± 0.007a 0.09 ± 0.006a
0.3 0.24 ± 0.019a 0.10 ± 0.008a 0.10 ± 0.007a
0.4 0.22 ± 0.029a 0.09 ± 0.009a 0.10 ± 0.005a
0.6 0.22 ± 0.007a 0.08 ± 0.018a 0.10 ± 0.007a
Mean values ± standard deviation denoted by the same letters within a column are
not significantly different at p P 0.05.
3.3. Application of the partition theory to the release of tryptophan,
riboflavin and peptides from whey microbeads
The equilibrium release values (Rmax) for tryptophan, riboflavin,
dipeptide and pentapeptide for the range of volume ratios studied
(0.13–0.6) are shown in Fig. 3. Partition theory can be used to
relate the affinity of compounds to the microbead or the release
medium. This theory (as described in Section 2.5) was applied to
the Rmax values of each of the compounds from the microbeads.
Tryptophan had the highest Rmax values of the four compounds
with 89 ± 1.5% released at a volume ratio of 0.13. However, this
value decreased with increasing volume ratio, with 57 ± 3%
released at a volume ratio of 0.6. A similar trend occurred for
riboflavin and the dipeptide with Rmax values decreasing with
increasing volume ratio, resulting in Rmax values of 41 ± 2.8% and
45 ± 3.6%, respectively at a volume ratio of 0.6. The pentapeptide
had significantly lower levels of release at equilibrium with Rmax
values ranging from 7.1 ± 0.3% to 3.1 ± 0.2% at volume ratios of
0.13 and 0.6, respectively.
The good fit of the model (solid line in Fig. 3) to the data strong-
ly suggests that release of the compounds is a partition driven dif-
fusion phenomenon, with the movement of the compounds out of
the microbeads due to diffusion to an extent determined by their
relative affinity to the protein bead. This suggests that the final
R% is influenced by interactions between the compounds and the
microbead. Hydrophobic interactions have previously been report-
ed as the driving force for partitioning (Wimley & White, 1993).
The partition coefficient (Krel) values for tryptophan, riboflavin,
dipeptide and pentapeptide partitioning between the microbead
and the release medium were 0.8 ± 0.17, 0.41 ± 0.03, 0.47 ± 0.06
and 0.027 ± 0.0062 respectively. These values were significantly
(p 6 0.05) different from each other.
Interestingly when the (Krel) values are compared to the capa-
city factor (k0 ) values it is evident that the more hydrophobic the
Pentapeptide molecule (indicated by a higher capacity factor k0 ), the lower the
kr (min1) equilibrium constant (Krel) and ultimately the lower the Rmax. The
0.014 ± 0.004a log of the (Krel) plotted against the log k0 (Fig. 4) showed a linear
0.011 ± 0.006a relationship. It appeared that the more hydrophobic the compound
0.017 ± 0.006a the stronger the interaction with the microbead, which resulted in
0.014 ± 0.003a
0.015 ± 0.003a
increased retention of the compounds in the microbead. Molina,
Rivarola, and Barbero (2012) reported that the equilibrium con-
stant (K) for the uptake of tryptophan and riboflavin into a range
of hydrogel polymers increased as the hydrophobicity of the poly-
mers increased. These authors reported that the interaction
between tryptophan and the hydrogels was by hydrophobic inter-
action through its aromatic rings. Furthermore, the authors also
discarded the effects of molecular weight by studying the parti-
tioning of compounds of similar molecular weight.
This suggests that whey microbeads could potentially be used
as an encapsulation system in high moisture functional foods and
beverages for compounds of a similar hydrophobicity to that of the
pentapeptide, as the diffusional losses at equilibrium are low. For
compounds that are less hydrophobic, such as riboflavin, the equi-
librium Rmax levels are too high to make the microbeads in their
current state a viable option for incorporation into beverages. A
previous study using whey/alginate beads reported that diffusional
losses of encapsulated riboflavin occurred within the shelf life of
the product, as a result the riboflavin would not be encapsulated
upon ingestion (Wichchukit, Oztop, McCarthy, & McCarthy,
2013). The results of the pentapeptide show that by establishing
an interaction between the compound and the microbead such
losses during the product’s shelf life could be largely overcome.
3.4. Mass balance between the kinetics of encapsulation and release
As both encapsulation and release kinetics followed pseudo first
order kinetics, it was expected that a directly proportional relation-
ship existed between the two processes. Examining the encapsula-
tion and release kinetics of tryptophan it was evident that as
volume ratio increased, the Emax increased and the Rmax decreases.
At a volume ratio of 0.2, the Emax was 19.6 ± 2.3% and Rmax was
82 ± 2.7%, giving a total of 101.6%, while at a volume ratio of 0.4
the Emax was 36.3 ± 1.5% and Rmax was 65 ± 1.4%, giving a total of
101.3%. Allowing for standard deviations the Emax and Rmax are
approximately equal to 100% across volume ratios. This shows

100 0.2

0.0

80 -0.2

-0.4

-0.6
Release %

60
log K rel

-0.8

40 -1.0

-1.2

20 -1.4

-1.6

0 -1.8
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.0 0.5 1.0 1.5
Vbead/Vaq log k'

Fig. 3. Release maximum (Rmax) values for tryptophan (d), riboflavin (s), dipeptide Fig. 4. Effect of hydrophobicity (as measured by capacity factor log k0 ) of trypto-
(N), and pentapeptide (4), at equilibrium as a function of volume ratio (Vbead/Vaq). phan (d), riboflavin (N), dipeptide (s), and pentapeptide (4)) on their respective
(Note: solid line represents the behaviour predicted by partition.) partitioning between an initial microbead phase and an aqueous phase.
 G.J. O’Neill et al. / Food Chemistry 180 (2015) 150–155
there is a complementary mass balance relationship between both
processes.
To improve on the accuracy of this calculation, changes in vol-
ume ratio were allowed for by using the equilibrium constant (K)
for encapsulation (Kencap) and release (Krel). The products of (Kencap)
and (Krel) for tryptophan, riboflavin, dipeptide and pentapeptide
were calculated as 1.05, 1.00, 1.09 and 0.99 respectively. The
results show an almost perfect correlation between the encapsula-
tion and release kinetics, which is indicated by a value of 1. This is
advantageous as when high levels of encapsulation are obtained
there will subsequently be low levels of diffusional losses, meaning
high levels of both encapsulation and retention of potentially valu-
able bioactive ingredients is achievable.
4. Conclusion
The results indicated that the volume ratio of microbead to
compound solution influenced the % encapsulation, although it
did not significantly influence the rate of encapsulation. However,
the rate of encapsulation did increase with increasing molecule
hydrophobicity. Loading of the microbeads was quite rapid, with
all four compounds reaching maximum uptake values in less than
40 min. This methodology is amenable to large scale manufacture,
as large batches of blank microbeads can be readily manufactured
and subsequently loaded in a short period of time with a range of
bioactive compounds. However, the release kinetics show that
release from the microbeads is also quite fast in the case of trypto-
phan, riboflavin and dipeptide. In contrast, results of the pentapep-
tide show that by establishing an interaction between the
microbead and bioactive that is sufficiently strong to minimise dif-
fusional losses, then whey microbeads have great potential as an
encapsulation system for small hydrophobic molecules in high
moisture foods.
Acknowledgement
The work described herein was supported by Enterprise Ireland
under Grant Number CC20080001.
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