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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
266
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),
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.
(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
100
100
99
99
85
90
83
88
3
4
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.
268
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.
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.
270
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.
271
Table 2
Mass of granules formed using various cations, following 21 min of repeated
impact testing
Cations
S.D. (%)
Ca2+
Ba2+
Fe3+
86
93
90
9
5
6
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. 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.
272