Enzyme and Microbial Technology 38 (2006) 265272

Granulation of subtilisin by internal gelation of alginate microspheres

for application in detergent formulation
Ariel W.J. Chan a , Isabelle Mazeaud b , Todd Becker b , Ronald J. Neufeld a,
a

Department of Chemical Engineering, Queens University, Kingston, Ont., Canada K7L 3N6
b Genencor International, 925 Page Mill Road, Palo Alto CA 94304, USA
Accepted 1 August 2005

Abstract
Subtilisin was encapsulated within impact-resistant alginate granules produced by emulsification, internal gelation, and acetone extractive drying.
The mechanical and controlled release properties of the granules were modified by adding to the alginate varying levels of formulation excipients,
including titanium dioxide, polyvinyl alcohol, microcrystalline cellulose, starch and sucrose. Optimum protease activity and mass yields of 83 and
88%, respectively (mg active subtilisin/g granules), occurred for granules formulated with 3% alginate, 10% starch, 10% titanium dioxide, and
3% subtilisin. Mass losses occurred primarily during the gelation step. Maximum encapsulation efficiency is achieved by using higher molecular
weight alginate, increasing the alginate concentration, and carefully controlling process temperature and pH. The strongest granules were obtained
at the higher concentrations of medium-G or high-G alginate, while fastest granule dissolution was achieved when a lower concentration of alginate
was used in combination with polyvinyl alcohol or microcrystalline cellulose as dispersants. Mechanical properties of alginate granules were found
to be unaffected by the different cations employed in matrix gel formation.
2005 Elsevier Inc. All rights reserved.
Keywords: Subtilisin; Internal gelation; Granulation

1. Introduction
Subtilisins are a class of alkaline serine endopeptidases with
molecular weights ranging from 25 to 35 kDa and isoelectric
point of 9.4 [1,2]. Because of broad specificity, subtilisins are
valuable to the detergent industry. Detergent formulations are
harsh environments since compounds such as bleach, alkyl benzene sulfonate and alkyl sulfate which are normally present,
can potentially denature the enzyme [3]. Moreover, proteases
are subjected to autoproteolytic degradation, particularly at elevated temperature and humidity, characteristic of storage in
consumer products such as detergents [3]. Therefore, protease
granulation within a protective matrix helps shield against harsh
environments and proteolysis. In addition, negative responses
to proteases such as asthmatic reactions [4], allergic-responses
or skin rashes [5] amongst exposed workers and consumers, are
minimized through granulation, combined with tighter engineering controls to reduce the airborne enzyme concentration.

Corresponding author. Tel.: +1 613 533 2785; fax: +1 613 533 6637.
E-mail address: neufeld@chee.queensu.ca (R.J. Neufeld).

0141-0229/$ see front matter 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2005.08.018

The objective of granulation, then, is to formulate dust-free

granules which disintegrate rapidly during the washing cycle,
releasing active subtilisin. In addition to the active enzyme,
additives which are normally incorporated into the formulation
include fillers/extenders, colorants, disintegrants, binders, lubricants and stabilizers, to achieve certain physical properties. In
general, the enzyme granules are expected to match the size
distribution and density of the bulk detergent base. This will
minimize the variability in washing performance and is desired
for aesthetic purposes.
Alginate is one of the most frequently used materials for
enzyme encapsulation, due to mild formulation conditions [6].
Alginate is a natural polysaccharide consisting of linear binary
copolymers of 14 glycosidically linked mannuronic acid (M)
and guluronic acid (G) residues. The relative amounts of the
two uronic acid monomers and the sequential arrangements vary
according to the origin of the alginate. Alginate is polyanionic
at physiological pH, and will form a three-dimensional gel network in the presence of divalent or multi-valent cations. Due
to spatial arrangement of the ring and hydroxyl oxygen atoms
of the monomers, poly-guluronate (GG blocks) form a stronger
ionic bonding with divalent cations, referred to as the egg-box

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model [7,8]. Therefore, the affinity with cations and the resultant gelling capacity is a direct function of the G-block size and
quantity, and the availability of cations.
Alginate can be gelled by internal release of the calcium,
through in situ gelation. The emulsification/internal gelation
method [9] was proposed for industrial scale production of
micron or sub-millimeter diameter beads. The sol containing
insoluble Ca-complex is dispersed in oil resulting in an alginate
sol in oil emulsion. By controlling emulsification conditions, the
bead mean size can be controlled [10]. The release of soluble
calcium ions from insoluble carbonate complex is triggered by
pH reduction from 7.5 to 6.5, inducing rapid gelation. Homogeneous alginate gels result in which calcium, alginate and enzyme
are uniformly distributed [11].
Prior research into alginate encapsulation has focused on its
use in the production of wet gel microspheres or beads. An
important commercial application is in the production of granulated active biologicals. The drying of gels is possible using
process equipment such as fluidized bed dryers, but there is very
little work on the production and properties of granulated hydrogels containing active biologicals. The granulation of subtilisin
in alginate was recently described by Liu et al. [12]. Granules
appeared spherical, discrete, and smooth, but improvements in
dusting levels, compromised dissolution and enzyme release
rates, and vice versa. The characterization of the effect of drying on the properties of the polymer matrix, such as granule
size, strength, permeability and release properties is essential
for commercial application. The present study was designed to
improve encapsulation yields while finding a good compromise
between granule mechanical strength and dissolution rate appropriate to detergent application. The effect of parameters such as
fractional alginate G-content, molecular weight, concentration,
gelling cations and additives on the physical properties of dry
granules was examined.
2. Materials and methods
2.1. Materials
Sodium alginates, SG150, SG300, SG500, S170, S550, Satialgine S1100X
and Algogel 3031 were kindly provided by Degussa Texturant Systems
(Boulogne Billancourt, France). Protonal SF102 was obtained from FMC
BioPoymer (Drammen, Norway). The chemical compositions and the average
molecular weights of these alginates were characterized by Nuclear Magnetic
Resonance and a multi-angle light scattering detector, respectively. The corresponding guluronic acid contents for SG150, SG300, SG500, S170, S550, S1100
X, Algogel 3031, and SF102 were determined to be 0.71, 0.60, 0.46, 0.37, 0.36,
0.37, 0.48, and 0.72, and the average molecular weights determined to be 278,
694, 800, 257, 666, 325, 369, 384 kg/mol, respectively.
For comparative purposes, commercial enzyme granules labeled sample 1
and sample 2 were provided by Genencor International. Both granule samples had an enzyme payload of 44.5 mg active subtilisin/g granules. Subtilisin
concentrate, Purafect UF (lot L-20031) concentrate with protein and activity concentration of 300 mg/mL and 92 mg active enzyme/mL, respectively
were manufactured by Genencor International (Palo Alto, USA). Detergent base
Gessy Lever and Ariel Futur were used in dissolution trials. TrisHCl buffer
consisted of 100 mM TrisHCl, 0.005% Tween 80, pH 8.6, and peptide substrate N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide (Vega Biochemicals,
Arizona, USA) was used in the activity assay.
Filler additives used in the formulation included polyvinyl alcohols of average molecular weight 95,000 (Sigma-Aldrich Canada Ltd. Oakville, Canada),

25,000 and 133,000 (Polysciences Inc., Warrington, USA), microcrystalline

cellulose (MCC) of average particle size 100 micron (MC-101 , Blanver
Farmoquimica LTDA. Sao Paulo, Brazil), carboxymethyl cellulose (CMC),
sodium starch glycolate (Blanver Farmoquimica LTDA, Sao Paulo, Brazil),
titanium dioxide (Dupont Inc., Palo Alto, USA), sucrose (Redpath Industries
Ltd., Toronto, Canada), corn starch (Best Foods Canada Inc., Canada), and
micro-sized calcium carbonate (Omya Inc., Orgon, France). Other chemicals
used included sodium carbonate, sorbitan monooleate and bovine serum albumin (Sigma-Aldrich Ltd., Oakville, Canada), Tween 80 and glacial acetic acid
(Fisher Scientific Ltd., Toronto, Canada), and canola oil (Canbra Ltd., Lethbridge, Canada).

2.2. Methods
2.2.1. Alginate microsphere formulation by emulsication/internal
gelation
Micron or sub-millimeter diameter alginate beads can be formulated by
emulsifying alginate sol in oil, followed by liberation of in situ Ca2+ from insoluble carbonate complex, giving rise to the gelation of the alginate microspheres
[9]. The so-called emulsification/internal gelation procedure is well suited to
large scale formulation of microspheres. As applied to subtilisin encapsulation
in the present study, alginate solution was prepared by dissolving sodium alginate in deionized water. Fillers/extenders were then dispersed in the alginate
solution as needed. The pH was adjusted to 56 and the slurry was then deaerated under vacuum for a few hours. Purafect UF concentrate was added into the
alginate sol formulation according to the payload desired. In order to avoid pregelation caused by calcium ions in the enzyme concentrate, sodium carbonate
was added into the Purafect concentrate at 1.5%. Ultrafine calcium carbonate
crystal suspension (10%), was sonicated for 5 min using an ultrasonic homogenizer (Cole-Parmer 4710 series) at 60% duty cycle, output control 6, then added
to the alginate slurry at a ratio of 0.1 (v/v of calcium carbonate suspension to
alginate slurry). The pH of the sol was monitored to ensure that it was in the
range of 77.5.
Alginate sol was emulsified in canola oil at a ratio of 1/4 (v/v of alginate sol
to oil) by mechanical mixer for 15 min. Sorbitan monooleate (Span 80) may be
also added to reduce the bead size.
The gelation step involved pH adjustment of the sol resulting in internal
liberation of the Ca2+ from carbonate complex in situ. Glacial acetic acid was
pre-dissolved in oil at a ratio of 0.1 (v/v of glacial acetic acid to oil), then added
to the emulsion to initiate gelation of the emulsified alginate droplets. Mixing
was continued for an additional 2 min.
The suspension was transferred at 1:1 volume ratio to CaCl2 solution
(50 mM) to facilitate gel microsphere partitioning into the aqueous phase. To
avoid microsphere aggregation, gentle mixing was provided in the oil phase at
approximately 80 rpm. After microspheres had partitioned into the lower aqueous phase, the residual oil supernatant was removed, and microspheres washed
with 0.5% Tween 80, then 50 mM CaCl2 to remove residual oil. For microspheres
containing subtilisin, the aqueous partitioning and washing steps were omitted.
Alternatively, direct vacuum filtration was used to prevent enzyme release from
the gel beads into the aqueous phase.
2.2.2. Alginate beads formulated using extrusion/external gelation method
Alginate solution of 13% (w/v) was mixed with various fillers/extenders
depending on the desired formulation. The slurry was then extruded via a droplet
generator (1000XL Automatic Liquid Dispenser, EFD, Canada) using syringe
needles (EFD, Canada) into a 100 mM capture solution containing Ba2+ , Ca2+ , or
Fe3+ . The bead diameter was controlled by the extrusion pressure, the concentric
air flow rate applied outside the syringe, and the internal diameter of the needle.
Although the gelation process should occur immediately when alginate sol was
in contact with the cation solution, fresh gel beads were further incubated in the
capture solution for 1 h to ensure complete gelation.
2.2.3. Acetone extractive drying
Freshly prepared microspheres were rinsed with acetone several times and
residual acetone was allowed to evaporate from the microspheres resulting in dry
granules, assisted if necessary by a gentle flow of air for 0.5 to 1 h. For microspheres containing enzyme, cold acetone at 4 C was used to prevent activity
loss.

A.W.J. Chan et al. / Enzyme and Microbial Technology 38 (2006) 265272

2.2.4. Liquefaction of alginate microspheres
Fresh alginate microspheres and dry granules were liquefied in 5% sodium
citrate TrisHCl buffer solution (w/v) at a ratio of 0.51% (w/v of beads to
solution) on an orbital shaker. The suspension was centrifuged after complete
microsphere or granule dissolution at 3600 g for 10 min. The resulting clear
supernatant phase was retained.
2.2.5. Determination of microsphere shrinkage during gelation and drying
Alginate sols of various compositions were extruded dropwise through a
syringe into 20 mL of oil containing 10% acetic acid (v/v). Forty gel beads were
collected and the bead shrinkage in mass was calculated by taking the mass
difference of the sol used and the gel formed. The mass difference of the dry
granules was also determined. These values were used for the estimation of the
enzyme concentration and protease activity in the fresh alginate microspheres
and dry granules.
2.2.6. Determination of protein concentration and protease activity
Protein concentration was determined spectrophotometrically at 280 nm [13]
(Molecular Devices Spectra Max 250, Alberville, USA).
Protease activity was monitored through the rate of formation of the colorific reaction product, p-nitroanilide, resulting from the cleavage of the amide
bond in the colorless peptide substrate, N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-pnitroanilide (suc-AAPF-pNA). This zero order kinetic reaction was measured
spectrophotometrically at 410 nm. The assay involved incubating 10 L sucAAPF-pNA, in 1 mL TrisHCl buffer solution at 25 C for 10 min prior to the
addition of a testing sample. Due to instrument sensitivity, testing samples were
diluted with TrisHCl buffer to around 0.04 mg active enzyme/mL. Absorbance
at 410 nm was taken at 0.33 s time intervals over 1 min. The maximum slope from
the time-absorbance profile was obtained to determine the enzymatic activity
using Eq. (1).
Activity (mg active subtilisin/mL) = slope dilution fold 0.052.

(1)

The activity was also expressed in international units (IU) defined as mol
of substrate hydrolyzed per min at 25 C and pH 8.6. The molar extinction
coefficient of p-nitroanilide is 8480 M1 cm1 .
2.2.7. Thermal stability of granulated enzyme
Test tubes (10 mL) containing either 5 mL fresh soluble or 1 g dry granulated
enzyme were incubated in a water bath at temperatures of 25, 30, 40, and 70 C
for various time increments. The relative activity was reported with respect to the
controls consisting of either fresh soluble subtilisin held at 4 C or dry enzyme
granules, held at 20 C. The baseline temperature of the controls for the two
sets of experiments were not expected to affect the comparison of the results, as
the enzyme is very stable at both temperatures, in either dry granular or soluble
form.
2.2.8. Subtilisin activity following extended storage
The long-term stability/shelf-life of granulated enzyme was examined by
placing 2 g granulated enzyme in sealed test tubes at room temperature. Three
samples were prepared and the activity was measured periodically.
2.2.9. Protein time release in detergent solution
Approximately 0.5 g granules were dispersed into 100 mL of 1% detergent
solution stirred by a 6-blade impeller at 300 rpm. At each sampling, 1 mL of
detergent solution was pipetted into a 1.5 mL eppendorf tube and centrifuged at
3400 g for 5 min, and protein concentration measured in the supernatant.
2.2.10. Repeated impact test
Mechanical strength of the enzyme granules was characterized by measuring
granule attrition and fragmentation dust produced in response to defined impact
forces, using the repeated impact test [14]. In the repeated impact test, granules
(0.5 g) with diameter 600710 m were placed inside a sealed steel chamber
having an inner dimension of 4.6 cm 3.3 cm 2.3 cm. The chamber was then
subjected to vibration with an amplitude of 1.6 cm and resonance frequency of
50 Hz. The impact strength was characterized as percentage of granules retained,
after sieving damaged granules and dust through a 300 m sieve.

267

2.2.11. Dissolution test

Dissolution time was determined by dispersing 0.5 g granules with diameter
range from 425 to 850 m, in 100 mL of 1% detergent solution, stirred by a 6blade impeller at 300 rpm and 20 C. Complete dissolution was observed when
the mixture passed through a 140 m sieve without leaving visible residual.
2.2.12. Data analysis
Three repeat experiments were carried out for most testing conditions, unless
otherwise specified. For protein concentration or enzymatic activity measurements, when the result is independent of time, three samples were obtained for
each repeated experiment to generate a sample mean and a sample standard
deviation. When the result is time-dependent, one sample was obtained at each
time interval and triplicate measurements were performed to generate a sample
mean and a standard deviation. The sample means and standard deviations were
later pooled to generate a pooled mean and standard deviation. The data points
presented in this work refer to the pooled means and standard deviations of the
corresponding data sets unless otherwise specified.

3. Results and discussion

3.1. Encapsulation yield
Subtilisin was encapsulated in a 3% SG150 alginate, 10%
TiO2 , and 10% starch mixture by emulsification/internal gelation. Enzyme concentrate was added to the formulation to
achieve a 3% payload (mg active subtilisin/g microspheres).
Encapsulation yields (activity and mass) for each step of the
encapsulation protocol are summarized in Table 1. The final payload was 86 mg protein/g granules or 5552 IU active enzyme/g
granules, which corresponds to 24 mg active enzyme/g granules. Overall, 83% of the initial enzyme activity, and 88% of
the protein mass, were retained in the final dry granules. The
main reduction in yield occurred in the gelation step (Step 3),
involving pH adjustment resulting in the liberation of soluble
Ca2+ from carbonate complex within the alginate sol droplet.
There are two possible explanations for this loss of both activity and protein during gelation. First, a significant amount of
gel water content was lost during this step, which accounted
for approximately 40% of the weight loss (data not shown).
Enzyme distributed close to the outer surface of the alginate
sol droplet may be expelled together with the water from the
shrinking droplets during gelation, resulting in the 10% loss of
encapsulated protein. Second, the enzyme may be subjected to
low pH microenvironments brought about by poor mixing and
Table 1
Subtilisin encapsulation efficiency at each production step
Step description

Activity yield (%)

Mass yield (%)

100

100

99

99

85

90

83

88

3
4

Encapsulants (TiO2 , starch,

subtilisin and calcium
carbonate) in alginate sol
Emulsion (alginate and
encapsulants mixed with
canola oil for 15 min)
Gelation step (pH adjustment
by addition of acetic acid)
Drying of fresh beads by
acetone extraction

The S.D.s of the activity and mass yields of five repeated experiments were less
than 8 and 10% of the mean values stated.

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distribution of the acid during pH adjustment, leading to an inactivation of some enzyme.

Subtilisin has broad pH stability (optimum between 7 and
8.5), yet is rapidly inactivated below pH 4 and above 11.5 [15].
Gelation is triggered by a pH reduction through the addition of
acetic acid to the emulsion. A small but quick reduction in pH
(typically from about 7.5 to 6.5) is desired to trigger the release
of Ca2+ from CaCO3 complex. The relatively high activity yield
observed in Table 1 was achieved through careful monitoring and
control of the initial pH prior to the triggering of the gelation
step, and by improved dispersion of the acid through adequate
mixing, in order to avoid localized zones of low pH.
A high viscosity of the oil can result in poor mixing and
thus dispersion of acid into the emulsion, leading to temporary
microenvironments of low pH. The converse is also true in that
certain microenvironments may not experience the desired pH
reduction, resulting in a partial release of Ca2+ within the sol,
and thus a slow rate of gelation. Shear within the emulsion can
deform the alginate droplets during gelation giving rise to nonspherical microspheres and granules. In addition, semi-gelled
microspheres aggregate during slow gelation as they continue
to be sticky in the pre-gel state.
Slow gelation may also occur if the initial pH of the sol is
elevated. For example, when the pH of the sol is initially above
8, poor gelation results in deformed particles as shown in Fig. 1a,
compared to the uniformly spherical appearance of the resulting
particles formed with a pre-gel pH of 7.5, as seen in Fig. 1b.
Hence, initial pH adjustment of alginate sol prior to gelation
is just as important as achieving a fast and effective dispersion
of the acid during gelation, leading to spherical and discrete
microspheres and subsequent granules.
3.2. Effect of alginate concentration and molecular weight
on the encapsulation yields
Alginate concentration and molecular weight has been found
to have a significant effect on protein retention or release from
the gel matrix [16,17]. The effects of alginate concentration and
its molecular weight on the final encapsulation yield at various
enzyme loadings are presented in Fig. 2. The final protein yield

Fig. 2. Protein mass yield in granules formulated with 2.3 (squares) or 3.9% (circles) alginate, and with high (closed symbols) or low molecular weight alginate
(open symbols), at various enzyme loadings. SG500 and Algogel alginates were
used, representative of high and low molecular weight alginates, respectively.

is seen to be dependent on both the enzyme loading and the composition of the polymer matrix. As enzyme loading increases, the
final mass yield decreases. A more dramatic mass yield change
is observed in granules formulated with a lower alginate concentration. The decrease in alginate concentration reduces alginate
cross-link density, increasing matrix porosity. Also, the increase
in enzyme loading may result in an increased distance between
the alginate junction zones, leading to a larger pore size, thus
higher permeability. In addition, it is seen that a higher molecular weight alginate enhances the encapsulation yield as seen in
Fig. 2. This may be explained by a decrease in matrix porosity,
resulting in higher protein retention during gelation. Lemoine
et al. [17] observed that the release rate of BSA from alginate
beads decreased as the molecular weight of alginate increased,
likely due to the decrease in alginate pore size. Kikuchi et al. [18]
suggested that the number of alginate cross-link points could be
increased by using a higher molecular weight alginate, leading
to a more densely cross-link polymer matrix, thus decreasing
the matrix porosity.

Fig. 1. Physical appearance of granules prepared using emulsification/internal gelation method. Granules were formulated using 3% SG150 alginate, 10% TiO2 , and
10% starch. The initial pH of alginate sol before gelation was 8.3 and 7.5 for granules in Fig. 2a and b, respectively. The bar shown represents a scale of 500 and
700 m in Fig. 2a and b, respectively.

A.W.J. Chan et al. / Enzyme and Microbial Technology 38 (2006) 265272

Fig. 3. Activity of dry granulated subtilisin incubated at 25 (), 30 (
), 40 (),

and 70 C () relative to dry granulated subtilisin stored at 20 C. All activity
assays performed at 25 C.

3.3. Properties of granulated subtilisin

The stability of granulated subtilisin at elevated temperatures is illustrated in Fig. 3. More than 80% of the activity of
the encapsulated subtilisin remained after 180 min at 70 C. In
comparison, free soluble subtilisin was 80% inactivated after
5 min at the same temperature (data not shown). Moreover, free
enzyme lost 20% of its activity within 10 min at 30 C, compared to the granulated enzyme which was fully stable for nearly
180 min at the same temperature. Granulated subtilisin demonstrated remarkable temperature stability in comparison to free
enzyme, over extended periods of time. A possible explanation
is that the dry alginate matrix may serve as a thermal insulator,
protecting enzyme from denaturation at elevated temperature.
In addition, Sharma and Gupta [19] found that the presence of
alginate in a heat-treated solution had a significant effect on
enhancing the thermal stability of lipases. It may be that alginate polymer effectively absorbs the heat, protecting the enzyme
from thermal inactivation.
The activity decay was examined over a 200-day period at
room temperature, as shown in Fig. 4 with the decay constants

Fig. 4. Activity decay profile of granulated subtilisin at room temperature.

269

Fig. 5. Effect of disintegrants, MCC (), PVA (
), sucrose (), and starch ()
on granule impact strength after 21 min of dusting test. The granules consisted
of 3% Algogel alginate, 10% TiO2 and various amounts of disintegrant loadings.
The impact strength of commercial sample 1 ( - ) and commercial sample 2
(- - -) is noted for comparison.

determined from the best fitted line, t1/2 = ln 2/k1/2 , of 0.0033

day1 .
3.4. Physical properties of alginate granules
The physical properties of alginate beads may be influenced
by parameters which include alginate concentration and composition, additives, and type of cation used for gelation [20,21].
Although these parameters are well understood for alginate gel
beads, there is little known about their affect on dry granules.
Granules were typically formulated with 3% alginate, 10%
TiO2 , and various amounts of disintegrants, which are intended
to aid in the dissolution or dispersion of the granule once in liquid
suspension. As can be seen in Fig. 5, granule strength decreased
as the amount of filler additives introduced to the formulation
increased. This may be due to the filler additives occupying
void spaces in the alginate matrix resulting in a decrease in
alginate chain interaction, subsequently leading to the formation of weaker granules, which become less resistant to dusting. Granules formulated using disintegrants other than starch,
with loading concentration less than 40% demonstrated greater
attrition resistance than the two commercial granular products,
samples 1 and 2. Granules containing microcrystalline cellulose
(MCC) were the least susceptible to dusting compared to the
other disintegrants tested. The difference in granule susceptibility to dusting may be due to the granule surface morphology,
which is largely affected by the types of fillers as shown in Fig. 6.
Granules without fillers appeared to be smoother compared to
those with filler additives with the exception of sucrose. Alginate/sucrose granules appeared smooth and slightly transparent.
Granules containing starch show a fluffy surface, which would
be more easily abraded resulting in a higher dust level.
Enzyme granules are designed to disintegrate during the
washing cycle to achieve rapid release of active enzyme. Dissolution time of granules containing different disintegrants is
presented in Fig. 7. When the loading concentration reached 5%,

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A.W.J. Chan et al. / Enzyme and Microbial Technology 38 (2006) 265272

Fig. 6. Granule morphology. Formulation consisted of 3% alginate alone or with 30% filler additives: MCC, PVA, starch, sucrose or 20% TiO2 . The granules shown
have a diameter of approximately 700 m.

the dissolution time for granules containing carboxymethyl cellulose (CMC) and sodium starch glycolate were 33 and 32 min,
respectively, which showed no noticeable improvement over
granules formulated without disintegrant. Reduction in dissolution time for the remaining disintegrants tested corresponded
with an increase in disintegrant loading. For instance, when the
MCC loading increased from 0 to 40%, the dissolution time
decreased from 36 to about 7 min. The commercial product,
sample 2 showed a dissolution time of less than 2 min. It is generally desired that the granules fully disperse within 15 min. The
most effective disintegrants were MCC and PVA.

Fig. 7. Effect of disintegrants, MCC (), PVA (
), sucrose (), and starch
() on granule dissolution time. The granules consisted of 3% Algogel alginate, 10% TiO2 and disintegrants at various levels of loading. The dissolution
time of commercial sample 1 (- - -) and commercial sample 2 (- -) were also
determined.

Among the granules compared in the dusting and dissolution

tests, MCC as a filler demonstrated the best combination of promoting rapid granule dissolution while having the least affect
on dusting property. If the required dissolution time is under
15 min, then 10% MCC loading would be appropriate (dissolution time of 14 min) while retaining excellent dusting properties
(93% intact granules after 21 min of repeated impact test).
3.5. Effect of alginate type on granule attrition resistance
and dissolution time
Granules were formulated with different alginates (3% concentration), containing 10% TiO2 and 30% MCC. The percentage of intact granules remaining following the dusting test, and
the dissolution times, are compared for different the alginates in
Fig. 8. Granules formulated with medium-G or high-G alginates
(>45% G-content) were much more stable during the dusting
test. Quong et al. [11] demonstrated that overall bead shrinkage during gelation is proportional to the amount of guluronic
acid content in alginate. This compaction of the gel structure
during gelation may improve the structural rigidity of alginate
matrix and thus result in stronger granules after drying. Granules formed using low-G alginates lost more than 60% of their
original mass to attrition during the dusting test. Any further
decrease in guluronic acid content is expected to result in the
formation of relatively weak granules.
Results of the dissolution test are compared for different alginates in Fig. 8. The differences in dissolution time were not
noticeable. Therefore, the guluronic acid content and possibly
the molecular weight of alginate play an important role in reinforcing the granule strength but not a significant role in affecting
the dissolution time.

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271

Table 2
Mass of granules formed using various cations, following 21 min of repeated
impact testing

Fig. 8. Effect of alginate composition on granule dissolution time (clear bars)

and percentage of intact granules (solid bars) following 20 min of dusting test.
The granules consisted of 3% alginate, 30% MCC, and 10% TiO2 . Data shown
are the means and S.D.s of a minimum of three repeated experiments.

3.6. Effect of alginate concentration on granule strength

and dissolution time
Granules were formulated with 10% TiO2 , 30% MCC
and different concentrations of alginate. Granule mechanical
strength was improved slightly by increasing the alginate concentration, as did the dissolution time, as seen in Fig. 9. A different detergent base was used in this experiment (Ariel Futur ),
whereas Gessy Lever was used in the experiment described in
Fig. 8. As different detergents contain different concentrations
of chelators and cations, the dissolution rate was different from
the previous data.

Cations

Mass remaining (%)

S.D. (%)

Ca2+
Ba2+
Fe3+

86
93
90

9
5
6

of alginate sol into a solution of the corresponding cation were

dried with acetone. Alginate sol consisted of 1.5% SG500 alginate, 20% sucrose, 10% starch, and 5% TiO2 , and the gelation
bath contained calcium, barium, or ferric chloride. There was no
appreciable difference in the mass retained following 21 min of
repeated impact test between granules formulated using different
cations, as seen in Table 2, suggesting that granule mechanical
strength was similar between the three types of granules. Smidsrod [22] showed that alginate gel strength increases as the size
of the cation increases. A possible explanation is that the stability of the polymer in hydrogels may depend mainly on the
ability of a cation to hold the neighboring polymer chains via
ionic attraction. However, the structure of dry granules is more
rigid which allows little or no movement between the polymer
chains. In this case, the structural stability of the granules may
be less affected by the type and amount of cations.
3.8. Release prole of subtilisin from dry granules into
detergent solution

The monovalent ions that are present in natural alginates are

displaced by calcium or other multivalent cations during gelation, resulting in the formation of a gel-like polymeric network
through ionic interaction. Beads prepared by droplet extrusion

The release profile of subtilisin from dry granules into detergent solution was examined to determine subtilisin release
kinetics under combined diffusion and granule disintegration.
Enzyme granules were formulated with 3% alginate, 10% TiO2 ,
and 40% MCC and 3% enzyme payload. The concentration of
soluble enzyme in the detergent solution during the dissolution
test was measured over a 15 min time course and was calculated
as percentage of the total enzyme payload in the granules. It was
observed that the enzyme was released rapidly into the detergent solution prior to the completion of granule disintegration

Fig. 9. Effect of alginate concentration on granule dissolution time (clear bars)

and percentage of intact granules (solid bars) following 21 min of repeated
impact test. The granules consisted of 3% SF102 alginate, 30% MCC, and 10%
TiO2 .

Fig. 10. Release profile of subtilisin from dry granules in 1% Gessy Lever
detergent solution. The formulation of the granules consisted of 3% Algogel
alginate, 10% TiO2 and 40% MCC.

3.7. Effect of cations on granule strength

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A.W.J. Chan et al. / Enzyme and Microbial Technology 38 (2006) 265272

as illustrated in Fig. 10. For instance, more than 50% of the

enzyme was freed from the granules in the first 30 s and close to
90% of the enzyme was released to the supernatant after 3 min
of dissolution test.
4. Summary
Granulation of active subtilisin was achieved through emulsification of alginate sol, followed by microsphere gelation
and acetone extractive drying. The resulting enzyme granules appeared discrete and spherical. Encapsulation yields were
increased with a higher alginate concentration and molecular
weight. Subtilisin losses during formulation can be accounted
for by the loss of water content during the gelation step and possible enzyme inactivation during pH adjustment. Overall, the
protein mass and activity yields were high, achieved through
appropriate process improvements.
The granule mechanical strength and the release properties
of the active enzyme are influenced by surface morphology and
granule structural integrity. Following 21 min repeated impact
testing, alginate granules containing no filler additives appeared
fully intact, and thus dust-free, whereas those with an increased
amount of fillers became more susceptibility to dusting, corresponding to a decrease in granule dissolution time. Granule
abrasion/attrition resistance can be enhanced with a higher alginate concentration and higher G-content alginate, while granule
dissolution rate is improved through a careful selection of filler
additives and loading levels. Alginate/microcrystalline cellulose
granules exhibit excellent mechanical strength, and rapid protein
release in detergent solution.
This study demonstrates the potential of using alginate as
a granulation material for quick release subtilisin, which with
appropriate filler additives, has potential for use in laundry detergent formulation.
Acknowledgement
The research was supported by the Natural Sciences and
Engineering Research Council of Canada.
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