Food Hydrocolloids 28 (2012) 20e27

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Microuidic production of monodisperse biopolymer particles with reproducible

morphology by kinetic control
Sophia Wassn a, c, *, Elisabeth Rondeau b, Kristin Sott a, d, Niklas Lorn a, c, Peter Fischer b,
Anne-Marie Hermansson a, c
a

SIK e The Swedish Institute for Food and Biotechnology, Box 5401, 402 29 Goteborg, Sweden
Institute of Food, Nutrition and Health, ETH Zurich, 8092 Zurich, Switzerland
Department of Applied Surface Chemistry, Chalmers University of Technology, 412 96 Goteborg, Sweden
d
Department of Chemical Engineering Design, Chalmers University of Technology, 412 96 Goteborg, Sweden
b
c

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 3 August 2011
Accepted 8 November 2011

Microdroplets of phase separating and gelling gelatin-maltodextrin mixtures were produced by

a microuidic technique. The microstructures observed inside the gelled particles were highly reproducible. This resulted from both the controlled production of monodisperse droplets in a microuidic
device and the tuning of gelation and phase separation kinetics.
We showed that the internal particle microstructure can be tailored by varying the cooling rate
(90
C/min, 55
C/min, 5
C/min), the biopolymer composition (4% gelatin and 6%e7.3% maltodextrin)
and the gelatin type (lime hide, pig skin). The particles were analyzed using confocal scanning laser
microscopy and image analysis. Microstructures with smaller domain sizes were formed at the fastest
cooling rate (90
C/min), and microstructures with large domain sizes were obtained at the slowest
cooling rate (5
C/min). Furthermore, differences in particle morphology were observed at this slowest
cooling rate. In particles containing pig skin gelatin, maltodextrin was located in the core, whereas
gelatin was present at the watereoil interface. The opposite was observed for particles consisting of lime
hide gelatin where the maltodextrin was found toward the oil phase. The results also showed that
a higher concentration of maltodextrin formed larger bicontinuous microstructures compared to the
ones obtained with lower concentrations.
2011 Elsevier Ltd. All rights reserved.

Keywords:
Microuidic
Phase separation
Gelation
Microparticles

1. Introduction
Microparticles as encapsulating vehicles for different substances
have gained great attention in the past decade (Burey, Bhandari,
Howes, & Gidley, 2008; Jones & McClements, 2010; Madene,
Jacquot, Scher, & Desobry, 2006; Theberge et al., 2010). Tailoring
the internal microstructure of microcapsules and microparticles
allows to designing and controlling their delivering properties, such
as preservation and targeted release of the encapsulated active
species (Tran, Benot, & Venier-Julienne, 2011; Yan et al., 1994).
Biopolymer microcapsules that can be used to encapsulate cells or
active species often consist of a hydrogel from a single biopolymer
such as alginate (Rondeau & Cooper-White, 2008; Yeh, Zhao, Lee, &
Lin, 2009; Zhang et al., 2006), k-carrageenan (Zhang et al., 2006),
chitosan (Tan, Choong, & Dass, 2009; Yang, Huang, & Chang, 2007) or
* Corresponding author. SIK e The Swedish Institute for Food and Biotechnology,
Box 5401, 402 29 Goteborg, Sweden. Tel.:46 105166648.
E-mail address: sophia.wassen@sik.se (S. Wassn).
0268-005X/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2011.11.004

gelatin (Huang et al., 2009). Few studies of microparticles made of

biopolymer mixtures are reported in the literature (Bulgarelli, Forni,
& Bernabei, 2000; Capretto, Mazzitelli, Luca, & Nastruzzi, 2010; Chen
& Subirade, 2006; Matalanis, Lesmes, Decker, & McClements, 2010;
Nochos, Douroumis, & Bouropoulos, 2008).
The release prole of the encapsulated substances has been
shown to be determined by the structure (Yan et al., 1994),
composition (Bulgarelli et al., 2000; Lee, Oh, Lee, & Lee, 2010) and
size properties of the microparticles (Sansdrap & Mos, 1993; Xu
et al., 2009). Xu et al. (2009) demonstrated that the release rates
from poly(lactic-co-glycolic acid) (PLGA) microparticles were
affected by the polydispersity and the size of the particles. They
showed that the drug release of bucivacaine was signicantly
slower from monodisperse microparticles produced by microuidics compared with microparticles obtained from conventional
emulsication with similar average size (Xu et al., 2009).
A controlled production of microparticles with perfectly reproducible sizes and morphologies is therefore essential for the
purpose of controlled delivery.

S. Wassn et al. / Food Hydrocolloids 28 (2012) 20e27

In the past few decades, the utilization of microuidic-based

techniques has rapidly developed and expanded. The controlled
generation of droplet-based systems using multiphase ow in
conned microchannels has been implemented in a wide variety of
applications. For droplet production, it is necessary that devices not
only produce a regular and stable monodisperse droplet stream but
that they are exible enough to provide droplets of a prescribed
volume at a prescribed ow rate (Baroud, Gallaire, & Dangla, 2010).
Three main approaches based on different physical mechanisms
have emerged: break-up in co-owing streams (Cramer, Fischer, &
Windhab, 2004; Guillot, Colin, Utada, & Adjari, 2007; Umbanhowar,
Prasad, & Weitz, 2000; Utada, Fernandez-Nieves, Stone, & Weitz,
2007), break-up in cross-owing streams (Christofer, Noharuddin,
Taylor, & Anna, 2008; Garstecki, Fuerstman, Stone, & Whitesides,
2006; van Steijn, Kreuzer, & Kleijn, 2007; Thorsen, Roberts,
Arnold, & Quake, 2001) and break-up in elongational strained
ows (Anna, Bontoux, & Stone, 2003; Dreyfus, Tabeling, & Willaime,
2003; Lee, Walker, & Anna, 2009). In all cases, the formation of
a uniform and continuous stream of evenly spaced droplets was
demonstrated and well-studied (Christofer & Anna, 2007; Engl,
Backov, & Panizza, 2008). Droplet polydispersity in such streams,
dened as the standard deviation of the size distribution divided by
the mean droplet size, is as small as 1e3% (Baroud et al., 2010). The
microuidics approach has been secured as a way to overcome the
limitations of conventional methods for droplet and particle
production. To the best of our knowledge, no microparticles consisting of segregative phase separating and gelling biopolymer
systems have been produced using a technique based on microuidics prior to this study.
Phase separating and gelling biopolymer mixtures of gelatinmaltodextrin were investigated in this work. They undergo segregative phase separation, generating separate regions enriched in
one biopolymer. The gelatin gelation kinetically traps the evolution
of the structure of the phase separation. The type of morphology
formed depends mainly on the phase volume of the biopolymer
system (Lorn, Altskr, & Hermansson, 2001; Norton & Frith, 2001;
Tolstoguzov, 2003). Discontinuous structures are formed when the
phase volumes are unequal, and the biopolymer with the lowest
phase volume will be dispersed into a continuous phase of the
other biopolymer. Bicontinuous structures are attained when the
phase volumes are equal or close to equal. The phase volume is
determined by the phase diagram, inuenced by biopolymer
concentration, temperature and solvent partition (Norton & Frith,
2001; Tolstoguzov, 2003). The nal microstructure is governed by
the kinetics of phase separation and gelation, and by the interplay
between the two processes which has been extensively studied in
bulk phase (Lorn et al., 2001; Lorn, Langton, & Hermansson, 1999;
Norton & Frith, 2001). The morphology of the gelatin-maltodextrin
system can therefore be designed according to composition and
kinetic conditions (Fransson, Peleg, Lorn, Hermansson, & Krger,
2010; Lorn et al., 2001).
We have previously demonstrated that gelatin-maltodextrin
mixtures can give rise to various morphologies inside gelled
microparticles and that the nal structure depends on the particle
size, biopolymer composition and quench temperature (Fransson,
Lorn, Altskr, & Hermansson, 2009; Fransson et al., 2010). Novel
microstructures, compared with the bulk phase, were found in
small particles with a diameter below 20 mm, showing that the
microstructure inside gelatin-maltodextrin microparticles was
affected by physical connement. Two types of microstructures
(core-shell or janus-like) were observed regardless of the thermodynamic conditions and biopolymer composition. A conventional emulsication method was used in these studies to allow
examination of microstructures in different sizes of microparticles
in one sample (Fransson et al., 2009; Fransson et al., 2010). Due to

21

the connement effects that have been discovered, producing

monodisperse particles appears to be equally as essential as
controlling the gelation and phase separation kinetics used to
obtain gelled microparticles with reproducible morphologies and
well-controlled properties.
The main objective of this work was to produce monodisperse
particles of a phase separating and gelling biopolymer mixture, and
to obtain a precise control of internal microstructures characterized
by confocal laser scanning microscopy. Designed microuidic
devices (MFDs) was utilized which allowed for the generation of
microdroplets with a well-dened size in the range of 115e160 mm.
To further demonstrate the possibility to design internal
morphologies different biopolymer compositions and process
conditions were used.
2. Material and methods
2.1. Materials
The biopolymers used were gelatin lime hide (LH) with Bloom
240 (Rousselot, Baupte, France), gelatin porcine skin (PS) with
Bloom 300 (SigmaeAldrich, St. Louis, USA) and Paselli SA2 maltodextrin (Avebe Group, Foxhol, Netherlands). The gelatin LH was
a pure rst extract with a number-average molecular weight of
83.8 kDa having an isoelectric point of pH 4.7. The gelatin PS was
a commercial gelatin with an isoelectric point between pH 7.0e9.0.
The maltodextrin was covalently labeled with Rhodamine B isothiocyanate (RITC) (De Belder & Granath, 1973; Garnier, Bourriot, &
Doublier, 1998; Lorn & Hermansson, 2000), making the sample
uorescent. Mixtures of gelatin and maltodextrin were used as the
dispersed phase (droplet phase). Rapeseed oil (Zeta, Di Luca & Di
Luca AB, Stockholm, Sweden) with 2% w/w of polyglycerol polyricinoleate (PGPR) (Danisco, Grindsted, Denmark) as emulsier was
used throughout the study as the continuous phase.
2.2. Design and fabrication of the microuidic devices (MFDs)
The microuidic channel network consists mainly of a sheath-ow
junction where the disperse phase meets the carrier phase and where
droplets are formed. A schematic of the microchannel pattern is
presented in Fig. 1a. The device design allows gentle mixing of the
biopolymer mixture (indicated as the Mixing Zone in Fig. 1a) that will
be processed upstream of the microdroplet creation site (indicated as
the Droplet Formation zone in Fig. 1a). This aims to ensure that the
composition of the disperse phase remains homogenous and
consistent over the whole experiment. Masters were prepared with
SU-8 photoresist (MicroChem, USA) in bas-relief on silicon wafers.
Microchannels were fabricated using a Dow Corning Sylgard Brand
184 silicone elastomer with a standard soft-lithography method,
which allows rapid replication of the integrated microchannel
prototypes. The microchannels were 100 mm deep.
2.2.1. Mixing zone (Mansur, Ye, Wang, & Dai, 2008; Tabeling, 2009)
Adequate mixing is a signicant challenge when dealing with
pressure driven laminar microows, where the Reynolds number is
so small that hydrodynamic instabilities and turbulences are
entirely suppressed. When two streams are injected side by side
into the device, mixing occurs only through diffusion. Splitting the
inlet streams into sub-streams and recombining them reduces the
size over which mixing must be achieved and increases the contact
surface between the two uids, causing diffusion to occur faster
and consequently reducing the mixing time. To further mix the
biopolymer solutions, we designed a mixing zone based on the
principle of producing thin liquid lamellae, typically in the range of
a few up to several tens of micrometers, and guided them through

22

S. Wassn et al. / Food Hydrocolloids 28 (2012) 20e27

Fig. 1. (a) Schematics of the microuidic ow-focusing geometry used for biopolymer droplet production indicating the Mixing Zone and the Droplet Formation zone. Channel
dimensions at the droplet formation zone for the ow-focusing (b) and the shear-focusing (c) geometries used in the present work.

a ow-through chamber (see Figs. 1 and 2 for more details). These

laminar-type mixers were inspired by a design described by (He,
Burke, Zhang, Zhang, & Regnier, 2001). The mixing zone was
introduced to preserve the one-phase mixture of gelatin and maltodextrin prior to manufacturing the droplets.

break-up, on the other hand. An extension of ow-focusing is

shear-focusing (Fig. 1c), which aims to create a region of highest
shear, existing in the contraction channel. As both designs operate
on the basis of the shearing of uids, a similar set of parameters
governs the dynamics of droplet generation.

2.2.2. Droplet formation

Biopolymeric droplets were generated by subjecting a stream of
the dispersed phase in the ow of the continuous phase via a owfocusing geometry. In a ow-focusing conguration (Fig. 1b), the
design employs symmetric shearing by the continuous phase on
the disperse phase. The geometry of the junction, together with the
ow rates and the physical properties of both phases (interfacial
tension, viscosities), determines the local ow eld, which in turn
deforms the interface and eventually leads to drop pinch off. The
size of the droplet is set by the competition between the pressure
caused by the external ow and the viscous shear stresses, on the
one hand, and the capillary pressure-resisting deformation and

2.3. Sample preparation

Fig. 2. Laminar passive mixers: Mixing is achieved with multiple intersecting channels
of varying lengths and a bimodal width distribution. The smaller channels are parallel
to the ow axis and vary in length, and the larger channels weave across the bed at
a 53
angle to the longitudinal ow axis. The bulk of the ow travels through the larger
channels and moves both longitudinally and laterally across the system during
transport through the bed.

Gelatin and maltodextrin were dissolved in distilled water with

slow agitation at 70
C for 40 min and at 95
C for 30 min,
respectively. The individual biopolymer solutions were mixed at
a weight ratio of 1:1. The nal gelatin concentration was kept
constant at 4% w/w, while the maltodextrin concentration varied in
the mixtures between 6 and 7.3% w/w. To avoid heat loss, the blend
was mixed in a water bath at 60
C and stirred for a few minutes
before it was loaded into the syringes.
2.4. Experimental set-up
To prevent the biopolymer mixture from phase separating and
gelling in an uncontrolled manner, all microuidic experiments
were done in a room with controlled temperature, set at 45
C
(5
C) and 22% rH humidity. Gas-tight microsyringes (S.G.E) were
lled with the liquids and mounted on motor driven syringe pumps
(PHD 2000 Harvard, Instech) that can synchronously operate two
syringes at various ow rates. The PDMS device was linked to the
dispenser syringes with polyethylene tubing (i.d. 0.58 mm;
o.d. 0.965 mm). Liquid biopolymer solution droplets in the
continuous phase owed from the device into a sample holder
prelled with heated oil via a segment of polyethylene tubing to
ensure that cooling was not initiated. Due to the temperature and
humidity conditions in the experimental chamber, it was not
possible to use a high speed camera linked to an inverted microscope to monitor the formation of droplets in the MFDs. The
formation of droplets was instead monitored using a DinoLite
camera, which captures 15 fps at a resolution of 1280  1024 pixels.
This still allowed control of the ow stability and was found to be
sufcient to ensure consistent droplet production. Biopolymer
droplets were collected in small steel cups after 10 min of stable

S. Wassn et al. / Food Hydrocolloids 28 (2012) 20e27

23

Fig. 3. CLSM micrographs showing microparticles of 4% w/w gelatin LH and 7.3% w/w maltodextrin-RITC (a) produced by mixing the oil and polymer mixture with a magnet stirrer
(b) produced by microuidics with ow rates of Qc 0.2 ml/hr and Qd 0.1 ml/hr. Both samples were quenched on a heat stage to 20
C with a cooling rate of 55
C/min. The scale
bars represent 100 mm in both images. Note that Fig. 3a consist of three merged images, taken at different heights in the same sample.

and constant ow through the MFDs. The sample was then cooled
from 45
C with different cooling rates of approximately 90
C/min
(in an ice bath), 55
C/min (on Linkam cooling stage) and 5
C/min
(in room temperature) to end temperatures of 4
C, 20
C or 25
C,
respectively, creating gelled biopolymer particles. All cooling rates
were estimated according to the average value of the temperature
decrease in the interval of 60
Ce25
C.
To clarify, in the following text non-gelled biopolymer droplets
are referred to as droplets while particles designates gelled
biopolymer droplets.
2.5. Confocal laser scanning microscope and image analysis
The confocal laser scanning microscopes (CLSM) utilized in this
work were a Leica TCP SP2 (Heidelberg, Germany) and a Leica TCS SP5
II (Heidelberg, Germany). The light source was an argon laser with an
emission maximum at 488 nm. The signal emitted in the wavelength
interval 530e680 nm was recorded. An HC PL APO immersion
objective with 20-time magnication and a numerical aperture (NA)
of 0.70, and a water objective with a 20-time magnication and a NA
of 0.50 were used throughout the study. Computer zooming between
1, 2 and 4 was done depending on the object acquired. The
images were recorded with 1024  1024 pixels. The CLSM images
were recorded well after the samples had reached the end temperature. The particle diameters were measured using image analysis
with CellSens Dimension 1.4 (Olympus Corp.).
3. Results and discussion
Microdroplets consisting of phase separating and gelling
gelatin-maltodextrin mixtures were generated in microuidic
devices. The size of the droplets was controlled by varying the ow
rates in the MFDs, and the internal microstructure of the gelled
biopolymer particles was precisely tuned by different temperature
cooling protocols (i.e. cooling kinetics) and varying both the
biopolymer concentration and its composition (i.e. gelatin type).
The nal internal structures of the particles were monitored using
confocal laser scanning microscopy (CLSM).
3.1. Formation of monodisperse droplets with reproducible internal
microstructure
The main goal of this study was to produce monodisperse particles
with reproducible internal microstructures under controlled

conditions. Conventional emulsication processes using rotor-stator

devices, homogenizers etc. give rise to relatively broad droplet size
distributions. As an example, Fig. 3a shows particles produced by
rapidly mixing oil and biopolymer solution together with a magnet
stirrer. This CLSM micrograph shows particles consisting of
a biopolymer mixture of 4% w/w gelatin LH and 7.3% w/w maltodextrin surrounded by the continuous rapeseed oil phase (seen as
black). Phase separation and gelation has occurred inside the particles, where gelatin is seen as the darker phase and maltodextrin
labeled with RITC is the brighter phase. Particles with a large polydispersity in particle diameter (below 5 mm to over 300 mm) were
found in the sample. The microstructures inside the particles varied,
and bicontinuous structures with a range of domain sizes were seen
together with particles displaying core-shell and janus-like structures. Fig. 3a illustrates the difculty, when using conventional
emulsication in the case of a sensitive phase separating mixture, to
prepare particles withholding a similar internal morphology.
However, reproducible internal morphologies have been produced
for less sensitive mixtures (Fransson et al., 2009; Fransson et al.,
2010); conventional emulsication has indeed allowed for the
investigation of phase separation and gelation in different connement sizes for biopolymer mixtures with the same concentration and
temperature history (Fransson et al., 2009; Fransson et al., 2010). It
should be noted that Fig. 3a consist of three different images put
together to illustrate the observed microstructures. All images were
taken in the same sample, but at different heights, since it was difcult to nd particles with different diameters in the same position.
To overcome size polydispersity inherent to conventional
emulsion methods, MFDs were designed and utilized. In these
devices droplets generated have a consistent size, and additionally
the temperatures of the mixture and of the ow cell itself are easily
tuned. Fig. 3b shows microparticles produced by microuidics with
ow rates of 0.2 ml/h for the continuous phase (Qc) and 0.1 ml/h for
the dispersed phase (Qd), in the case of a biopolymer mixture of 4%
w/w gelatin LH and 7.3% w/w maltodextrin. The bicontinuous
Table 1
Average particle diameter at different ow rates for the dispersed phase (Qd) and the
continuous phase (Qc).
Biopolymer

Qd
(ml/hr)

Qc
(ml/hr)

Average diameter
(mm)

Polydispersity

4 w/w% gelatin PS
4 w/w% gelatin LH
4 w/w% gelatin LH

0.1
0.1
0.03

0.2
0.2
0.35

149.9  8.8
161.4  10.9
117.1  8.4

5.87%
6.75%
7.17%

24

S. Wassn et al. / Food Hydrocolloids 28 (2012) 20e27

Fig. 4. CLSM micrographs showing biopolymer particles of 4% w/w gelatin PS and 7.3% w/w maltodextrin-RITC from two different batches, produced by microuidics. The ow rates
were Qc 0.2 ml/hr and Qd 0.1 ml/hr. Both samples were quenched on a heat stage from 45
C to 20
C with a cooling rate of 55
C/min. The scale bars represent 100 mm in both
images.

microstructure seen inside the microparticles is similar for all

particles in the sample, as well as the domain sizes. This illustrates
the reproducibility of the internal microstructures in particles
produced in a microuidic device.
For a given gelatin-maltodextrin mixture, smaller droplets are
generated when increasing the ow rate of the continuous phase,
which increases the frequency of the droplet production, for a given
volume ow rate of the dispersed phase. The average diameter of
the particles are shown in Table 1, as well as the size polydispersity
(dened as the ratio between the standard deviation and the
average diameter of the particles multiplied by 100), determined

from captured images of more than 200 droplets. The polydispersity varied between 5.9 and 7.2% for all biopolymer mixtures
investigated, which represents not an extremely narrow but
a tolerable size distribution. This size polydispersity can be the
consequence of different factors: coalescence between non-gelled
droplets and break-up during droplet collection can lead to particles with different sizes, even thought droplets are monodisperse. It
can also result from the particle size measurements themselves, as
the CLSM images might sometimes be taken slightly above or
below the absolute middle of a particle. For the present work, most
important is that these variations in size are small enough not to

Fig. 5. CLSM micrographs showing biopolymer particles with varying cooling rates and end temperatures of two different gelatin types (aec) 4% w/w gelatin LH and (dee) 4% w/w
gelatin PS. The maltodextrin concentration is constant at 7.3% w/w (a, d) 90
C/min cooling rate from 45
C to 4
C, (b, e) 55
C/min cooling rate from 45
C to 20
C and (c, f) 5
C/
minute cooling rate from 45
C to 25
C. The ow rates were Qc 0.2 ml/hr and Qd 0.1 ml/hr. The scale bars represent 100 mm in all images.

S. Wassn et al. / Food Hydrocolloids 28 (2012) 20e27

induce any signicant changes in the particle internal

microstructure.
It was also critical to conrm that identical particle microstructures are obtained in the case of different production batches.
Fig. 4a and b show two CLSM micrographs of biopolymer particles
from two different samples. Both mixtures consist of 4% w/w
gelatin PS and 7.3% w/w maltodextrin-RITC and were produced at
the same ow rates and cooling prole.
The bicontinuous microstructure inside the microparticles is
seen for all particles in the sample, and the microstructures formed
were very similar for both experiments. This conrmed that
production of droplets in a microuidic device, coupled with
temperature control allows for the generation of reproducible
microstructures inside phase separated biopolymer particles. Note
that the biopolymer mixture was seen to be stable in the syringes
up to several hours, as long as they were at a temperature above the
phase separating temperature (Lorn et al., 2001) (results not
shown).
The possibility to generate droplets monodisperse in size and at
the same time control the internal morphology allows for a reproducible production of capsules with desired functionalities.
3.2. Kinetic control of the microstructure
The morphology of the particles produced with microuidics
can be tuned by applying temperature cooling protocols (referred

25

to as kinetic control), corresponding to different cooling rates and

end temperatures. Fig. 5 shows particles consisting of 4% w/w
gelatin and 7.3% w/w maltodextrin-RITC generated using three
different cooling rates, in the case of two different gelatin types
(LH and PS), and resulting from droplets all generated at the same
ow rates.
As described in the introduction section, the end morphology in
bulk phase separation is determined by the initiation and interplay
between phase separation and gelation (Lorn et al., 1999). A fast
cooling rate (90
C/min) increased the gelation rate, interfering
with the phase separation process. Gelation kinetically arrested the
spinodal decomposition at an early state in the evolution, and
resulted in a bicontinuous structure with small domain sizes, as
shown in Fig. 5a and d. A slower cooling rate of 55
C/min (Fig. 5b
and e) allowed the phase separation process to evolve further due
to a longer time before gelation interfered resulting in bicontinuous
structures with considerably larger domain sizes inside the particles than in those with a faster cooling rate. With an even slower
cooling rate (5
C/min), the phase separation process could develop
longer and the biopolymers separated almost completely. The
phase separation has reached a state in Fig. 5c and f at which the
domain sizes are very large. These results show that the nal
microstructures inside the particles, in the same way as in a bulk
phase, can be directed by kinetic control. At the same time reproducibility within the particle populations is maintained when
droplets are generated in microuidic devices.

Fig. 6. CLSM micrographs showing biopolymer particles with different biopolymer concentrations: (a) 6% w/w maltodextrin and 4% w/w gelatin LH, (b) 7.3% w/w maltodextrin and
4% w/w gelatin LH, (c) 6% w/w maltodextrin and 4% w/w gelatin PS and (d) 7.3% w/w maltodextrin and 4% w/w gelatin PS. The ow rates were (a) Qc 0.35 ml/hr and Qd 0.03 ml/
hr, (bed) Qc 0.2 ml/hr and Qd 0.1 ml/hr. All samples were quenched on a heat stage from 45
C to 20
C with a cooling rate of 55
C/min. The scale bars represent 100 mm in all
images.

26

S. Wassn et al. / Food Hydrocolloids 28 (2012) 20e27

Fig. 7. CLSM micrographs showing biopolymer particles with different gelatin types: (a) gelatin LH (b) gelatin PS. The biopolymer concentration is constant at 4% w/w gelatin and
7.3% w/w maltodextrin in both samples. The ow rates were Qc 0.2 ml/hr and Qd 0.1 ml/hr. Both samples were cooled from 45
C to 25
C with a cooling rate of 5
C/min. The
scale bars represent 50 mm in both images.

3.3. Effect of biopolymer concentration and composition

The internal microstructure of the particles can also be altered
by changing the biopolymer concentrations and compositions. This
is illustrated in Fig. 6: Fig. 6a and b show two different concentrations of maltodextrin mixed with gelatin LH, both cooled from
45
C to 20
C at 55
C/min. Particles with the lower concentration
of maltodextrin, 6% w/w (Fig. 6a), have discontinuous microstructures, with maltodextrin inclusions in the continuous gelatin phase
(Fransson et al., 2009; Fransson et al., 2010). Particles with a higher
maltodextrin concentration e 7.3% w/w e (Fig. 6b) have a bicontinuous internal microstructure. An increase in the maltodextrin
concentration makes the phase volume in the system vary, and
consequently, the morphology shifts from a discontinuous to
a bicontinuous microstructure (Lorn et al., 2001; Norton & Frith,
2001; Tolstoguzov, 2003).
In addition, Fig. 6a and b illustrates the inuence of the
continuous ow rate on the droplet size. Small droplet diameters
can be produced from higher ow rates in the microuidic channels, i.e. in the droplet formation zone (see Fig. 1) (Anna et al., 2003;
Thorsen et al., 2001). The average particle diameter in Fig. 6a was
117.1 mm and in Fig. 6b 161.4 mm owing to a faster ow rate in the
former case, seen in Table 1.
The trend of smaller microstructures at lower concentrations of
maltodextrin was also found in the mixtures with gelatin PS. This is
seen in Fig. 6c and d. Bicontinuous structures were observed for 6%
w/w maltodextrin (Fig. 6c) and 7.3% w/w maltodextrin (Fig. 6d),
and higher concentrations of maltodextrin resulted in larger
bicontinuous structures. In contrast to gelatin LH, an increase in the
maltodextrin concentration did not change the type of microstructure. This might be the result of water distributing differently
in the two types of gelatin used in this study (Tolstoguzov, 2003).
Differences in microstructure between the particles with gelatin
LH and PS were found to be more pronounced at the lowest cooling
rate of 5
C/min (as previously discussed in section 3.2), shown in
Fig. 7.
Fig. 7a shows particles prepared using gelatin LH. A distinct layer
of maltodextrin can be seen in the outermost part of the particles,
while gelatin is found mainly in the core of the particles. These results
agree with those of previous studies (Fransson et al., 2009; Fransson
et al., 2010). Fig. 7b shows a sample consisting of 4% w/w gelatin PS
and 7.3% w/w maltodextrin. Gelatin PS is depleted from the core of the
microparticles but enriched at the surface of the particle, i.e. toward

the oil phase (the black phase in the CLSM micrographs). In contrast
to the gelatin LH-maltodextrin mixture, maltodextrin is seen closer to
the center of the particles than in the case of mixtures of gelatin PSmaltodextrin. No signicant differences in the microstructure could
be visually determined with the CLSM at this length scale for the two
faster cooling rates comparing the particle microstructures in Fig. 5a
with 5d, and Fig. 5b with 5e. It should be noticed that the two gelatins
have different isoelectric points and the observations made may
possibly be an effect of the different characters and charges of the two
gelatins types. This could result in different behaviors in multiphase
systems and could also affect the afnity toward the oil-emulsier
phase used in this work. Another possible reason is a difference in
interfacial tension between the oil-emulsier phase and the three
biopolymers used. In a previous study by Lorn et al. (1999) different
gelatin types were observed to affect the microstructures in bulk
phase, although only the size of the maltodextrin inclusions were
investigated. The actual mechanism for the different appearance
inside the particles is still unclear, but the underlying mechanisms are
currently being investigated.
4. Conclusions
Monodisperse microdroplets of a phase separating and gelling
biopolymer mixture with well-dened sizes ranging from
approximately 115e160 mm were produced using a microuidic
device. In a subsequent step, the kinetics of gelation and phase
separation of the system was tuned by controlling the cooling rate.
The microstructures obtained were highly reproducible within one
particle population and among different batches with the same
composition, size and cooling prole. The internal morphology of
the particles, i.e. the domain sizes and type of microstructure, was
determined by the cooling proles, biopolymer concentrations and
types of biopolymers (LH and PS gelatin).
These particles could have potential uses in food products and
pharmaceuticals since the materials are food grade. Scaling-up the
production of microparticles may be feasible by parallelization of
the microuidic devices. We speculate that structured particles
with different morphologies such as core-shell, discontinuous, and
bicontinuous may give rise to different release rates. Internal
particle structure in relation to release rates has previously been
shown to be important (Yan et al., 1994). Future works will involve
determination of mass transport in phase separating biopolymer
mixtures with different microstructures, in which the molecular

S. Wassn et al. / Food Hydrocolloids 28 (2012) 20e27

diffusion rates in the two phases are considerably different. Local

mass transport can be measured with FRAP (Fluorescent recovery
after photo bleaching) (Braeckmans, Peeters, Sanders, Smedt, &
Demeester, 2003). Recently a new evaluation model, rectangleFRAP, has been developed which gives even higher precision
when measuring local diffusion in heterogeneous materials
(Deschout et al., 2010). This concept may open up new possibilities
for controlled release of active substances by smart design of the
internal microstructure of particles.
Acknowledgments
All the authors acknowledge the nancial support of the European Commission within the Sixth Framework program (NMP3-CT2006-033339: Controlled Release e New controlled release
systems, produced by self-assembly of biopolymers and colloidal
particles at uideuid interfaces). For nancial support the
Swedish Research Council, VR, is also acknowledged. Many thanks
to Erich J. Windhab for numerous discussions during the course of
the investigation.
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