Food Sci. Biotechnol.

24(5): 1735-1739 (2015)

DOI 10.1007/s10068-015-0225-6

Research Note

Microencapsulation of Catechin with High Loading and

Encapsulation Efficiencies Using Soaking Methods
Eun Suh Kim, Ji-Soo Lee, and Hyeon Gyu Lee*
Department of Food and Nutrition, Hanyang University, Seoul 04763, Korea

Received December 29 2014

Revised April 29 2015
Accepted April 29 2015
Published online October 31 2015
*Corresponding Author
Tel: +82-2-2220-1202
Fax: +82-2-2281-8285
E-mail: hyeonlee@hanyang.ac.kr
pISSN 1226-7708
eISSN 2092-6456
© KoSFoST and Springer 2015
Abstract Catechin-loaded calcium alginate microparticles with high encapsulation and loading
efficiencies were prepared using two types of soaking methods; swelling and absorption methods. The
soaking methods, respectively, showed approximately 2.4× and 21.7× higher encapsulation and
loading efficiencies than the conventional method. Compared with the swelling method, the
absorption method showed significantly (p<0.05) higher encapsulation and loading efficiencies that
were controlled in the range of 10.6-51.6 and 4.9-38.2%, respectively, under different preparation
conditions, including alginate viscosity, blank particle quantity, and the catechin concentration. The
absorption method also showed better sustained release in simulated gastric and intestinal fluids and a
smaller particle size with uniform morphological properties than the swelling method. The absorption
method is a promising method for microencapsulation of catechin.

Keywords: catechin, calcium alginate microparticle, soaking method, encapsulation efficiency, loading
efficiency

Introduction
Catechins, polyphenolic compounds classified as flavan-3-ols of the
flavonoid group, are known to exert health-promoting antioxidant,
anti-inflammatory, and antitumor effects (1). However, there are
only a few instances where catechin can be applied as a functional
material due to a short half-life in plasma and instability in neutral
and alkaline intestinal environments (2). Therefore, protection of
catechins from the external environment is needed and encapsulation
techniques have been studied as a solution.
Biopolymeric delivery systems have been reported to increase
bioavailability and stability under unfavorable light, oxygen, and
temperature conditions (3,4). The natural polysaccharide alginate
has been used as a suitable coating material due to good biocompatibility,
non-toxicity, and stability in gastric and degradation in intestinal fluid
(5,6). An alginate-derived oral delivery system has been conventionally
prepared based on dropping a mixture of core material and alginate
into a CaCl2 solution (7,8). In previous studies, Lactobacillus spp. and
α-tocopherol were encapsulated in alginate gel particles to improve
stability and bioavailability (9,10). However, low encapsulation efficiency
of water-soluble substances, such as catechin, timolol maleate, and
ascorbic acid has been indicated as problems (11).
Encapsulation efficiency is an important property for evaluation of
oral delivery systems (12). Therefore, in order to improve the
encapsulation efficiency, double coated calcium pectinate gel beads
using liposomes (13), an internal gelation method using liquid paraffin
(14), and chitosan microparticles using the emulsion technique with
cyclomethicone and cyclopentasiloxane as emulsifiers (15) were
investigated. These methods improved the efficiency over the
conventional method; however, a complicated process and use of
toxic organic materials chloroform and paraffin limit use of the
method in food applications (16).
In the conventional method, core materials are encapsulated
using gelation of a polysaccharide containing core materials with a
multivalent cation, such as calcium. However, in the soaking methods,
blank particles were prepared in the same way as the conventional
method without core materials first, then particles were soaked in
aqueous core materials destined for encapsulatiion (17). Methylene
blue and 4-phenylazoaniline, which represented water soluble and
insoluble materials, respectively, were encapsulated effectively and
release behaviors were also controlled using the soaking method
(18). In addition, the soaking method is regarded as simple, effective,
and safe because toxic solvents are not used in processing. For these
reasons, the soaking method is viewed as promising to overcome a
low encapsulation efficiency and fast release of catechin.
The aim of this study was to investigate the encapsulation and
loading efficiencies of catechin-loaded microparticles using both the
conventional and soaking methods. Furthermore, the influence of
different preparation conditions for the soaking method on encapsulation
and loading efficiencies, particle size, morphology, and in vitro
1736 Kim et al.
release behavior, were investigated.
Materials and Methods
Materials Low (250 cp in a 2% (w/v) solution at 25oC) and high
(350 cp in a 1% solution at 20oC) viscosity sodium alginate was
purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and
Kanto Chemical Co. (Kyoto, Japan), respectively. Calcium chloride was
purchased from Yakuri Chemicals Co. (Kyoto, Japan) and catechin was
supplied by Unigen Co. (Cheonan, Korea). Other chemicals were
reagent grade and used without further purification.
Preparation of catechin-loaded microparticles Catechin-loaded
microparticles (MPs) were prepared based on the conventional (CM),
swelling (SM), and absorption methods (AM) following a modified
protocol of Tu et al. (18). For CM, a mixture of 3% (w/w) sodium
alginate and 10 mg/mL catechin was sprayed into a 4% (w/v) CaCl2
solution using an air atomizing system (151BL-6L; Spraying System
Co., Incheon, Korea). Resulting MPs were collected using a sieve (325
mesh) after hardening for 15 min at room temperature followed by
lyophilization (FD8508; Ilshin Co., Seoul, Korea).
For soaking methods, 3% (w/w) sodium alginate solution without
catechin was sprayed into 4% (w/v) CaCl2 solution under the same
conditions as for CM and, after 15 min of hardening, blank MPs were
collected using the sieve. For AM, blank MPs (3.5, 7, 10.5, and 14 g)
were suspended in 30 mL of a catechin solution dissolved in 30%
(w/w) ethanol with 100 rpm of stirring (MS-MP8; Daihan Co., Seoul
Korea) for 1-8 h, then centrifuged (Combi 408; Hanil Co., Inchon,
Korea) at 11,180×g for 10 min. For SM, lyophilized blank MPs of
identical weights (0.25, 0.5, 0.75, and 1 g), which were calculated
from the particle weight before and after drying, were added to a
catechin solution. The resulting suspensions were stirred and
centrifuged under the same conditions as for AM. The difference
between SM and AM MPs was the entrapment step in which dry
(lyophilized) blank MPs were used for SM while wet (unlyophilized)
blank MPs were used for AM. Collected catechin-loaded SM and AM
MPs were lyophilized and stored for subsequent evaluation.
Encapsulation and loading efficiencies Catechin-loaded MPs (100
mg) were placed in 30 mL of a 50% (w/w) methanol solution containing
0.1% (w/w) H3PO4 with 100 rpm of stirring for 12 h followed by
20 min of sonication(JAC 1505; Jinwoo Co., Seoul, Korea) (14). An
aliquot of 70 µL of the aqueous solution containing the extracted
catechin was mixed with 0.7 mL of 0.1% (w/v) 4-dimethylamino-
cinnamaldehyde (DMACA) in methanol/HCl 9:1 (v/v). The mixture
was vortexed (G-560; Scientific Industries, Bohemia, NY, USA) and
centrifuged at 2,000×g for 1 min. The resulting supernatant was kept
for 6 min at room temperature, then the absorbance was measured
at 622 nm using a UV spectrophotometer (Biomate 3S; Thermo
Scientific, Madison, WI, USA) (19). Catechin encapsulation (EE) and
Food Sci. Biotechnol.
loading efficiencies (LE) were determined as:
Actual amount of catechin entrapped in particles
EE (%)= ×100 (1)
Theoretical amount of catechin entrapped in particles
Actual amount of catechin entrapped in particles
LE (%)= ×100 (2)
Weight of particles
In vitro release studies Simulated gastric (SGF) and intestinal fluid
(SIF) were prepared using 0.05 M sodium chloride adjusted to pH 1.5
with HCl and 0.05 M sodium dihydrogen phosphate buffer adjusted
to pH 6.8 with NaOH, respectively (20). Catechin-loaded MPs were
immersed in the simulated digestive solutions at 37oC with 100 rpm
of shaking (J-USR; Jisico Co., Seoul Korea) and collected at 30, 60,
120, and 240 min. Each sample of dispersed microparticles was
centrifuged at 2,000×g for 5 min, and the amount of released
catechin in the supernatant was determined as described above. The
release ratio of catechin was determined as (21):
Release ratio (%)
Amount of catechin released from particles
= ×100 (3)
Amount of catechin initially entrapped in particles
Physical properties Particle size analysis was carried out in an
aqueous solution using a laser diffraction particle size analyzer
(Mastersizer S; Malvern, Worcestershire, UK). The morphology of
catechin-loaded MPs was observed using field-emission scanning
electron microscopy (FE-SEM, JSM-6330F; Jeol Korea Ltd., Seoul,
Korea). Samples were mounted on a metal stub using carbon tape
and coated with an approximate 30 nm thickness of gold. Electron
micrographs were taken at a magnification of 1,500×.
Statistical analysis All experiments were performed in triplicate. All
data were expressed as a mean value±standard deviation (SD).
Significant differences between mean values were determined using
an independent samples t-test and a one way analysis of variance
(ANOVA), followed by Duncan’s multiple comparison test (SPSS
12.0.1; SPSS Inc., Chicago, IL, USA).
Results and Discussion
Encapsulation and loading efficiencies Catechin-loaded MPs
prepared using the soaking method with 4% low viscosity alginate,
3% CaCl2, and 10 mg/mL of catechin resulted in EE and LE values of
2.45-30.96 and 6.20-13.83%, respectively. However, CM MPs prepared
under similar preparation conditions showed an EE value of 10.4%
and an LE value of 0.6% (Fig. 1A). Therefore, catechin-loaded MPs
prepared using the soaking method had much higher EE and LE
values than CM MPs. EE and LE values based on the soaking method
were mostly not affected by the soaking time significantly (p<0.05).
The stability of catechin was monitored during the loading step and
the amount of catechin remained stable for 8 h.
 Catechin Encapsulation Using the Soaking Method 1737

An increase in the quantity of soaked blank MPs resulted in a

significant (p<0.05) increase in EE values and a significant (p<0.05)
decrease in LE values, compared with controls. The EE value, which is
the ratio of the actual entrapped catechin vs. the initial amount of
catechin used, increased with an increase in the quantity of blank
MPs in which the catechin could be entrapped. However, core
materials were absorbed into blank MPs by passive diffusion in the
soaking method (22) and the increase in the quantity of blank MPs
under identical catechin concentrations decreased the catechin
concentration gradient across the wall of blank MPs. Therefore, the
LE value, the ratio of the actual amount of catechin entrapped vs. the
weight of MPs, decreased with an increase in the quantity of blank
MPs.
AM MPs showed higher EE values (10.65-30.96%) and LE values
(13.83%) than SM MPs (2.41-8.52 and 8.40%, respectively). Differences
in efficiency between the 2 particle types could be attributed to use
of unlyophilized blank MPs for the AM encapsulation process,
whereas lyophilized particles were used for SM. The freeze-drying
step can increase the porosity of the gel structure (23); however,
dried gel particles do not swell sufficiently in an ethanol solution
during the soaking process of SM MPs (17). Therefore, the diffused
catechin inside the SM MPs leaked more easily during the soaking
process than for AM MPs.
With an increase in alginate viscosity, the LE value of AM MPs
significantly (p<0.05) decreased from approximately 10.68 to 4.89%,
however, the EE value significantly (p<0.05) increased from
approximately 30.96 to 50.79% (Fig. 1B). Higher alginate viscosities
produced a denser gel structure with an increased cross-linking
strength that probably inhibited absorption of catechin into blank
MPs during the soaking process (24,25). However, blank MPs
prepared using low viscosity alginate were easily damaged during
the soaking and lyophilizing processes. As a result, approximately
1.25× fewer low viscosity alginate particles than high viscosity Fig. 1. Encapsulation and loading efficiencies of SM and AM MPs with
alginate particles were collected after the soaking process, resulting different amounts of blank microparticles. (A), different alginate
viscosities (B), and different catechin concentrations of AM MPs (C).
in a decrease in the EE value. Means with different letters differ significantly (p<0.05).
An increase in the catechin concentration (10-130 mg/mL) resulted
in a significant (p<0.05) increase in the LE value of AM MPs from 4.89
to 38.22% (Fig. 1C). The passive diffusion that led to entrapment of slowed due to a denser gel network structure formed at a higher
catechin in blank MPs continued until the catechin concentration alginate concentration (Figs. 2A and 2B) (17). However, this tendency
gradient across the walls of blank MPs reached equilibrium (22). was not observed for catechin release from SM MPs in SIF probably
Therefore, the amount of entrapped catechin increased with an because SM MPs degraded quickly with no control on catechin
increase in the catechin concentration gradient. However, in spite of release in SIF. Thus, MPs prepared using 3% alginate for both AM and
an increased catechin concentration, the EE value remained approximately SM, which showed the least catechin release, were used for further
50% with no change, probably because the catechin gradient evaluation.
reached equilibrium at a 50% EE value and, thus, entrapment of AM MPs showed a lower release rate than SM MPs in both SGF
catechin in blank MPs by passive diffusion ceased. and SIF (Fig. 2C). AM MPs showed an approximate 55-57% release of
catechin, whereas SM MPs showed an approximate 77-92% release
In vitro release properties Catechin release from SM and AM MPs in SGF and SIG after 1 h. SM MPs had a more porous structure than
in simulated digestive fluids significantly (p<0.05) decreased with an AM MPs due to a 2× freeze-drying step. Therefore, the more porous
increase in the CaCl2 concentration at fixed alginate concentrations structure allowed for an increased surface area with more interaction
of 3% (w/w) and catechin (30 mg/mL) because release of catechin between MPs and the dissolution medium, resulting in an increase in

October 2015 | Vol. 24 | No. 5

1738 Kim et al.
Fig. 2. Catechin release ratios with different CaCl2 concentrations for
SGF (A) and SIF (B) after 1 h, and fractional catechin release from SM
and AM MPs in SGF and SIF (C). Means with different letters and
asterisks differ significantly (p<0.05).
the contents released. However, both AM and SM MPs showed an
initial burst of catechin release in SGF and SIF over 30 min, perhaps
also due to the porous structure of alginate MPs that readily
admitted water and allowed diffusion. Burst release of a core
material is one of the major problems in encapsulation. Therefore,
further study is needed to reduce the burst effect.
Particle size AM MPs had a slightly smaller size and a better size
distribution than SM MPs (Fig. 3A). SM MPs prepared by soaking 0.5
and 1 g of blank MPs showed mean sizes of 130.6 and 157.7 µm, and
AM MPs prepared by soaking 7 g and 14 g of blank MPs showed
mean sizes of 40.3 and 114.4 µm, respectively, due to aggregation of
MPs that contributed to an increased particle size during the
Food Sci. Biotechnol.
Fig. 3. Size distributions (A) and SEM images showing the morphology
of SM MPs (B) and AM MPs (C).
lyophilization process. For this reason, SM MPs, which required
freeze-drying 2× during the encapsulation process, were larger than
AM MPs.
Morphology SM MPs showed a rugged form, while AM MPs
showed a better size uniformity (Figs. 3B and 3C). This difference in
appearance was caused by aggregation during the lyophilization
process. AM MPs were smaller than SM MPs, based on SEM analysis,
similar to results for size analysis. Particle morphologies were not
affected by differences in the quantity of soaked blank MPs for either
AM or SM.
In conclusion, the soaking method is a promising method to increase
EE and LE values over the conventional method of encapsulation.
Both EE and LE values were influenced by the quantity of soaked
blank MPs and the alginate viscosity. Catechin release from
microparticles decreased with an increase in the CaCl2 concentration.
AM MPs showed higher EE and LE values and lower levels of catechin
release than SM MPs. In addition, AM resulted in a smaller particle
size with better morphologic properties. Therefore, AM MPs were
regarded as more appropriate for encapsulation of low Mw food
ingredients like catechin, than SM MPs.
Acknowledgments This work was supported by the Technological
Innovation Research and Development Program (S1071836) funded
by the Small and Medium Business Administration (SMBA), Republic
of Korea.
Disclosure The authors declare no conflict of interest.

References
1. Kim HS, Quon MJ, Kim J. New insights into the mechanisms of polyphenols
beyond antioxidant properties; Lessons from the green tea polyphenol,
epigallocatechin 3-gallate. Redox Biol. 2: 187-195 (2014)
2. Clifford MN, van der Hooft JJ, Crozier A. Human studies on the absorption,
distribution, metabolism, and excretion of tea polyphenols. Am. J. Clin. Nutr.
98: 1619S-1630S (2013)
3. Lima AC, Sher P, Mano JF. Production methodologies of polymeric and
hydrogel particles for drug delivery applications. Expert Opin. Drug Del. 9:
231-248 (2012)
4. Fang Z, Bhandari B. Encapsulation of polyphenols-A review. Trends Food Sci.
Tech. 21: 510-523 (2010)
5. Lertsutthiwong P, Noomun K, Jongaroonngamsang N, Rojsitthisak P,
Nimmannit U. Preparation of alginate nanocapsules containing turmeric oil.
Carbohyd. Polym. 74: 209-214 (2008)
6. Chan AW, Neufeld RJ. Modeling the controllable pH-responsive swelling and
pore size of networked alginate based biomaterials. Biomaterials 30: 6119-
6129 (2009)
7. Jain D, Bar-Shalom D. Alginate drug delivery systems: Application in context of
pharmaceutical and biomedical research. Drug Dev. Ind. Pharm. 40: 1576-
1584 (2014)
8. Li Y, Hu M, Du Y, Xiao H, McClements DJ. Control of lipase digestibility of
emulsified lipids by encapsulation within calcium alginate beads. Food
Hydrocolloid. 25: 122-130 (2011)
9. Chandramouli V, Kailasapathy K, Peiris P, Jones M. An improved method of
microencapsulation and its evaluation to protect Lactobacillus spp. in
simulated gastric conditions. J. Microbiol. Meth. 56: 27-35 (2004)
10. Yoo SH, Song YB, Chang PS, Lee HG. Microencapsulation of α-tocopherol using
sodium alginate and its controlled release properties. Int. J. Biol. Macromol.
38: 25-30 (2006)
11. Sezer A, Akbuga J. Release characteristics of chitosan treated alginate beads:
II. Sustained release of a low molecular drug from chitosan treated alginate
beads. J. Microencapsul. 16: 687-696 (1999)
12. Hassannejad Z, Khosroshahi M, Firouzi M. Fabrication and characterization of
magnetoplasmonic liposome carriers. Nanosci. Technol. 1: 1-9 (2014)
Catechin Encapsulation Using the Soaking Method 1739
13. Lee J-S, Chung D, Lee HG. Optimization of calcium pectinate gel beads for
sustained-release of catechin using response surface methodology. Int. J. Biol.
Macromol. 42: 340-347 (2008)
14. Lee J-S, Kim EJ, Chung D, Lee HG. Characteristics and antioxidant activity of
catechin-loaded calcium pectinate gel beads prepared by internal gelation.
Colloid. Surface. B 74: 17-22 (2009)
15. Elabbadi A, Jeckelmann N, Haefliger OP, Ouali L. Complexation/encapsulation
of green tea polyphenols in mixed calcium carbonate and phosphate micro-
particles. J. Microencapsul. 28: 1-9 (2011)
16. Chen L, Remondetto GE, Subirade M. Food protein-based materials as
nutraceutical delivery systems. Trends Food Sci. Tech. 17: 272-283 (2006)
17. Sriamornsak P, Nunthanid J, Cheewatanakornkool K, Manchun S. Effect of
drug loading method on drug content and drug release from calcium
pectinate gel beads. AAPS PharmSciTech 11: 1315-1319 (2010)
18. Tu J, Bolla S, Barr J, Miedema J, Li X, Jasti B. Alginate microparticles prepared
by spray-coagulation method: Preparation, drug loading and release
characterization. Int. J. Pharm. 303: 171-181 (2005)
19. Dalluge JJ, Nelson BC. Determination of tea catechins. J. Chromatogr. A 881:
411-424 (2000)
20. Cacace J, Reilly EE, Amann A. Comparison of the dissolution of metaxalone
tablets (Skelaxin) using USP apparatus 2 and 3. AAPS PharmSciTech 5: 29-31
(2004)
21. Lee J-S, Chung D, Lee HG. Preparation and characterization of calcium
pectinate gel beads entrapping catechin-loaded liposomes. Int. J. Biol.
Macromol. 42: 178-184 (2008)
22. Sriamornsak P, Kennedy RA. Effect of a small molecule on diffusion and
swelling properties of selected polysaccharide gel beads. Carbohyd. Polym.
79: 219-223 (2010)
23. Yamamoto T, Nishimura T, Suzuki T, Tamon H. Control of mesoporosity of
carbon gels prepared by sol-gel polycondensation and freeze drying. J. Non-
Cryst. Solids 288: 46-55 (2001)
24. Mukhopadhyay D, Saville D, Tucker IG. Crosslinking of drug-alginate granules.
Part 2. Effect of granule preparation and composition on granule properties.
Int. J. Pharm. 356: 193-199 (2008)
25. Chan LW, Jin Y, Heng PWS. Cross-linking mechanisms of calcium and zinc in
production of alginate microspheres. Int. J. Pharm. 242: 255-258 (2002)
October 2015 | Vol. 24 | No. 5