Journal of Biomaterials and Nanobiotechnology, 2011, 2, 347-352
doi:10.4236/jbnb.2011.24043 Published Online October 2011 (http://www.SciRP.org/journal/jbnb)
347
Chitosan Sub-Micron Particles Prepared Using
Sulfate Ion Salt as Bacteriostatic Materials in
Neutral pH Condition
Kanako Saita1,2, Shoji Nagaoka2,3*, Maki Horikawa2,3, Tomohiro Shirosaki2,3, Shigeki Matsuda2,
Hirotaka Ihara1,3*
1Department of Applied Chemistry and Biochemistry, Kumamoto University, Kumamoto, Japan; 2Kumamoto Industrial Research
Institute, Kumamoto, Japan; 3Kumamoto Institute for Photo-Electro Organics, Kumamoto, Japan.
Received June 4th, 2011; revised July 22nd, 2011; accepted September 5th, 2011.
ABSTRACT
In this paper, the newly developed ion exchange phase separation method to create chitosan sub-micron particles is
introduced: 1) chitosan was dissolved in a lactic acid aqueous solution; 2) the obtained chitosan solution was added
stepwise in a sodium sulfate aqueous solution and cooled down to 5˚C to become slightly turbid through agglutination;
3) desalinating and deacidifying of the mixture was carried out by a dialyzing tube method. IR spectroscopy and ele-
mental analysis indicated that the agglutination of chitosan was induced by crosslinking effect with an electrostatic
interaction between sulfate anions and amino groups in the glucosamine unit although large excess of Na2SO4 caused
undesirable further agglutination of the resultant chitosan particles. As a result, the proper amount of Na2SO4 was ap-
proximately 1.0 - 10.0 equivalent for the amino group to create the chitosan particles with a sub-micron size. In addi-
tion, we investigated an antibacterial activity test for Escherichia coli of the obtained chitosan particles. The significant
antibacterial activity was observed in incubation even at neutral pH condition while the chitosan microbeads (size: ca
200
 m) prepared by the conventional method and chitosan granules (size: ca 600  m) as starting materials showed
almost no antibacterial activity in the same condition.
Keywords: Chitosan, Particle, Crosslinking, Microbeads, E. coli
1. Introduction
Chitosan is a cationic biopolymer obtained from N-
deacetylation of chitin, β-(1,4)-N-acetyl-D-glycan [1].
The non-toxic, biocompatible and biodegradable proper-
ties of chitosan provide potential for many types of ap-
plications [2-4]. Chitosan and derivatives have become
useful polysaccharides in the biomedical field. Especially,
these microparticles have been utilized as chroma-
tographic packings [5,6], enzyme-immobilized support
[7,8], affinity adsorbents for proteins [9], endotoxin ad-
sorbents [10] and dr ug carriers [11,12]. Generally, it w as
popular to use chitosan as antibacterial compounds in
agriculture, as elicitors of plant defense responses [13],
as additives in the food industry, as flocculating agents
for wastewater [14] and as pharmaceutical agents in
biomedicine [15,16]. In this content, environmentally
antibacterial activity of chitosan has received consider-
able attention recently. However, these activities are li-
mited to acidic conditions because of its poor solubility
above pH 6.5, where chitosan starts to lose its cationic
nature [17-20].
Chitosan is generally insoluble under a neutral pH
conditions because of a strong hydrogen bonding and
lower pKa (ca. 6) of a residual amino group. Thus, the
molding, investigation and application of chitosan have
been restricted. The methods for producing porous and
spherical chitosan microbeads, such as the “suspension
evaporation method” [6] and the “suspension crosslink-
ing technique” [9,21] using chitosan acid aqueous solu-
tions, have been reported. These methods require the use
of organic solvents and emulsifier. Also a sphering me-
thod by sp ray drying [22 ,23] is known and wid ely appli-
cable. However, these methods have some disadvantages:
for example, the particle size control is difficult, espe-
cially in below tens of micron but also a heating process
as a cost up factor is necessary. Recently, the “rapid ex-
pansion of supercritical fluid technology” [24] has been

Chitosan Sub-Micron Particles Prepared Using Sulfate Ion Salt as Bacteriostatic Materials in Neutral pH Condition 348
developed as a method preparing sub-micron particles.
Since this technique uses supercritical CO2, the process
is environmentally safe but it is also known that the co n-
trol of particle size, shape and composition are difficult
[25].
In this paper, we introduce a new method as the “ion
exchange phase separation method” to create submicron
particles from chitosan without using any organic emul-
sifier and solvent as well as with no heating process. This
method can be characterized by solidification with
Na2SO4 and following dialysis. It is also reported that the
obtained chitosan particles shows excellent antibacterial
activity even at neutral pH 7 towards Escherichia coli
although chitosan exhibits higher antibacterial activity
only in an acidic medium [26,27].
2. Materials and Methods
Chitosan sub-micron particles were prepared as follows:
two kinds of chitosan materials (CS85 and CS485) were
selected. Their chitosans are 85 mol% in the deacetyla-
tion degr ee, and w ere 70 - 100 kD a and 440 - 530 kD a in
the molecular weight, respectively. Chitosan was dis-
solved in a 1.3 wt% lactic acid aqueous solution to be 1.5
wt% and then 20 ml of the solution was added stepwise
into Na2SO4 aqueous solutions (1.0, 2.0, 5.0 and 10.0
equivalents for the amino group in a glucosamine unit) at
30˚C. The mixture was cooled down to 5˚C and kept for
10 min to become turbid. The mixture was desalinated
and deacidified by dialyzing with a dialysis tube
(MWCO, 12000 - 14000, Spectra/por 5) in distilled wa-
ter for 4 days. Th e obtained chitosan particles are abbre-
viated as PCS85-sm and PCS485-sm.
The other type of chitosan spherical microbeads was
prepared by slight modification of the suspension evapo-
ration method reported previously [6]. 25 ml of a 1.5
wt% chitosan (CS85 or CS485) lactic acid solution was
added to 250 ml of decahydronaphthalene containing 5 g
of polyethylene glycol mono-4-nonylphenyl ether (po-
lymerization degree, 10) and suspended by stirring at
80˚C for 24 h. By gradual removal of water, the chito-
san-containing suspension particles were solidified to be
spherical. The obtained microbeads were deacidified
with 1 M NaOH, and successively washed with ethanol
and ether. The obtained chitosan particles are abbrevi-
ated as PCS85-m and PCS485-m.
Surface area analysis of the particles was carried out
by the Brunau-Emmet Teller (BET) method using Auto-
sorb-1 (Aionics Co. Ltd., Japan). Fourier transformed in-
frared (FT-IR) spectroscopy was carried out with JASCO
FT/IR-700. The particle size and distribution were meas-
ured by dynamic light scattering (DLS) method (Zetasiz-
ernano-ZS, Sysmex Corp., Japan). The particles were
also observed using a field emission scanning electron
microscope (FE-SEM) (S-4000, Hitachi, Co. Ltd., Japan)
and stereomicroscope (KH-7700S, Hirox Co. Ltd., Ja-
pan).
For assay of antibacterial activity, Mueller-Hinton
Broth (Becton Dickinson and Company, USA) adjusted
by cation, which contained 92 mg of CaCl22H2O, 104.5
mg of MgCl26H2O and 15 g of agar for 1l of broth, re-
spectively, was used as culture media. Escherichia coli
NBRC 3972 (E. coli) was incubated at 37˚C for 4 - 6 h in
the Mueller-Hinton Broth until logarithmic phase was
reached. The assay was carried out with 0, 0.5, 1.0 and
5.0 mg/ml of the chitosan sub-micron particles (PCS85-
sm and PCS485-sm), the chitosan microbeads (PCS85-m
and PCS485-m) and ch itosan granules as starting materi-
als (abbreviated as CS85-g and CS485-g). The chitosans
were put into sterile glass-plates, 15 ml of Mueller-Hin-
ton Broth was added to each sterile glass-plate contain-
ing E. coli culture and then were spread on the plates to
be 1.0 × 102, 1.0 × 104 and 1.0 × 106 cfu/plate, and these
were incubated at 37˚C for 18 h. Antibacterial activity
was observed by comparison of with the control plate
without chitosan.
3. Results and Discussion
3.1. Preparation of Chitosan Sub-Micron
Particles
Figure 1 shows the FT-IR spectra of the product pre-
pared by the ion exchange phase separation method from
CS85. As shown in Figure 1(a), the product before dia-
lyzing showed a distinct adsorption at 1700 cm–1 corre-
sponding to C=O of a carboxyl group of lactic acid. The
adsorptions of S=O at 1110 cm–1 and S=O at 620 cm-1
increased with increasing amount of Na2SO4 added in the
sphering process. This indicates that the product was the
mixture of Na2SO4, chitosan and lactic acid. After dia-
lyzation for 4 days, the resultant product sho wed that the
adsorption at 1700 cm–1 for C=O disappeared completely
but also the adsorptions based on S = O at 1110 cm–1 and
S = O at 620 cm–1 decreased remarkably. However, S=O
at 1110 cm–1 and S = O at 620 cm–1 of the products re-
mained regardless of sufficient dialyzing. Similar IR
spectrum was observed for the product prepared from
CS485. These results indicate that large excesses of lac-
tic acid and Na2SO4 could be removed by dialysis, but
that anion-exchange from a lactate ion to a sulfate ion
towards an ammonium ion of chitosan occurred. The
final products, PCS85-sm and PCS485-sm were insolu-
ble in water.
3.2. Microscopic Observation of Chitosan
Particles
In the ion exchange phase separation method, a chitosan
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Chitosan Sub-Micron Particles Prepared Using Sulfate Ion Salt as Bacteriostatic Materials in Neutral pH Condition349
Figure 1. FT-IR spectra of PCS85-sm and PCS485-sm. a)
before and b) after desalinating. Added amou nt of Na2SO4:
0, 1.0, 2.0, 5.0, 10.0 (eq.) for -NH2 group.
lactic acid aqueous solution becomes turbid gradually by
addition of Na2SO4 and cooling down to 5˚C. The turbid-
ity remained after removing excesses of lactic acid and
Na2SO4 by dialysis. On the other hand, similar turbidity
increase was observed when a monoanion salt such as
NaCl was used instead of Na2SO4 but the dialysis made
it clear. This significant difference between Na2SO4 and
NaCl can be attributed to a divalent property based on
SO42– because IR spectroscopy showed a distinct absorp-
tion based on S=O even after successive dialysis as
shown in Figure 1. Therefore, it is estimated that SO42–
works as a sort of a crosslinker.
Stereomicroscopic images of PCS85-sm and PCS485-
sm in aqueous dispersions are shown in Figures 2 and 3,
respectively. The particle size was affected by the
amount of Na2SO4 used in the sphering process espe-
cially before dialysis. It is clearly shown that the agglu-
tination of chitosan particles was promoted with increase
of the amount of Na2SO4 and extreme agglutination was
observed in PCS485 as shown in Figure 3(a). This is due
to the fact that increase of amino groups as crosslinking
sites in chitosan promotes agglutination among chitosan
particles. As a result, the aggregate size reached over 50
Figure 2. Stereomicroscope images of PCS85-sm in disper-
sion after desalinating. 0, 1.0, 2.0, 5.0, 10.0 stand for added
amount of Na2SO4 (eq.) for -NH2 group.
Figure 3. Stereomicroscope images of PCS485-sm in disper-
sion after desalinating. 0, 1.0, 2.0, 5.0, 10.0 stand for added
amount of Na2SO4 (eq.) for -NH2 group.
m in PCS485-sm and to 5 - 10 m in PCS85-sm by ag-
glutination. Therefore, the sub-micron particles without
agglutination can be produced by a proper combination
of the concentration of Na2SO4 and the polymerization
degree of material chitosan.
On the other hand, the PCS485-m microbeads pre-
pared by the suspension evaporation method showed a
typical spherical shape and the particle size was about
200 - 300 m as shown in the SEM image of Figure 4(a).
Figure 4(b) showed the SEM image of material chitosan
granules (CS485-g, ca 600 m).
3.3. Size Distribution of Chitosan Particles in
Dispersions
As shown in Figure 5, the DLS dia grams show ed a cou-
ple of peaks in both the PCS85-sm and PCS485-sm dis-

Chitosan Sub-Micron Particles Prepared Using Sulfate Ion Salt as Bacteriostatic Materials in Neutral pH Condition 350
Figure 4. SEM images of PCS-m (a) and PCS-g (b).
Figure 5. Size distribution of PCS85-sm (a) and PCS485-sm
(b) before desalinating, determined by DLS. Added amoun t
of Na2SO4 for -NH2 group: 1.0 eq.
persions prepared with 1.0 eq. of Na2SO4 before dialyz-
ing. Their Z-averages were 1.06 m and 1.46 m, re-
spectively. This indicates that both the dispersions in-
clude sub-micron size particles but also agglutination of
the sub-micron particles occurs somewhat. The aggluti-
nation was promoted with increase of Na2SO4 used in the
preparation procedure as mentioned in the microscopic
observation. As supporting this, the Z-average of the
samples prepared with 2.0 or higher eq . of Na2SO4 could
not be obtained because the detection limit of DLS was
within 10 m and thus these samples might produce ex-
tremely large aggregates. On the other hand, to remove
excess of lactic acid as well as Na2SO4 by dialysis sup-
pressed this agglutination distinctly. For example, the
Z-averages of the PCS85-sm dispersions after dialysis
were estimated to be 0.97, 1.34, 2.80 and 4.24 m in the
preparation with 1.0, 2.0, 5.0 and 10 eq. of Na2SO4, re-
spectively.
However, the peaks indicated in Figure 6(a) showed
smaller particle sizes than those Z-averages. This indi-
cates that agglutination was not completely suppressed
by dialysis.
3.4. Antibacterial Activity for Chitosan Particles
We investigated the antibacterial activity of the obtained
chitosan particles for E. coli. PCS85-sm prepared with
1.0 eq. of Na2SO4 was selected for this purpose because
the particle agglutination was most suppressed compar-
ing with the others. Also PCS85-m and CS85-g were
used for comparison.
The first application was carried out at pH 5.0 because
it is known that usual chitosan exhibits antibacterial ac-
tivity only in an acidic medium such as pH 5.4 - 6.5 [26,
Figure 6. Size distribution of PCS85-sm (a) and PCS485-sm
(b) after desalinating, determined by DLS. 0, 1.0, 2.0, 5.0,
10.0 stand for added amount of Na2SO4 (eq.) for -NH2 group.
27]. Figure 7(a ) shows the growth of E. coli in the pres-
ence of CS85-g. The sample preparation is as follows:
CS85-g was dispersed to be 0.5 mg/ml, 1.0 mg/ml and
5.0 mg/ml in an incubation medium at pH 5.0 adjusted
with a 0.2 M HCl aqueous solution. After the given
amount of agar was added to each dispersion, E. coli
culture was spread on the above-mentioned media. As
expected, CS85-g showed significantly antibacterial ac-
tivity with the increase of its concentration. On the other
hand, when an incubation medium was adjusted at a-
round pH 7.0 with a 0.2 M NaOH aqueous solution and
then E. coli culture were spread and incubated with the
same procedure, distinct growth of E. coli was observed
even in the presence of 5.0 mg/ml of CS85-g as shown in
Figure 7(b). This indicates that CS85-g showed almost
no effect in inhibition of growth of E. coli under neutral
condition. Similar no inhibition at pH 7.0 in E. coli
growth was observed in PCS85-m prepared by the sus-
pension evaporation method as shown in Figure 7(c).
Complete inhibition of E. coli growth at pH 7.0 was
observed only in the presence of PCS85-sm prepared by
the ion exchange phase separation method. Figure 7(d)
shows the typical example. No E. coli colony was found
for 48 h incubation even in the low concentration of 5.0
mg/ml. The mechanism of growth inhibition fo r E. coli is
not yet specified but it is consid ered presumably that the
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Chitosan Sub-Micron Particles Prepared Using Sulfate Ion Salt as Bacteriostatic Materials in Neutral pH Condition351
Figure 7. Growth of E. coli in culture containing PCS-g in
pH5.0 (a) PCS-g in neutral condition (b) PCS85-m in neu-
tral condition (c) and PCS-sm in neutral condition (d).
size effect may be included but also the specific surface
area of the particles can play an important role. For ex-
ample, the specific surface areas were determined by
BET method to be 127 m2/g, 15.19 m2/g, and 2.82 m2/g
in PCS85-sm, PCS85-m and CS85-g, respectively. It is
reasonable to consider that increase of the surface area
promotes the adsorption of E. coli onto the chitosan par-
ticles and thus effective antibacterial activity can be de-
rived from amino groups of chitosan.
4. Conclusions
The chitosan submicron particles have been prepared by
the newly developed ion exchange phase separation
method which is characterized by the facts that chitosan
is dissolved in a lactic acid aqueous solution and solidi-
fication can be realized by addition of Na2SO4 and cool-
ing down to 5 ˚C. By removal of excess of lactic acid and
Na2SO4, particle agglutination can be sufficiently sup-
pressed. Probably a stable dispersion state in water can
be attributed to crosslinking effect between amino groups
of chitosan and sulfate anion from Na2SO4.
In addition, antibacterial activity for E. coli has been
investigated. It was conf irmed that only the chitosan sub-
micron particles prepared by the present method showed
distinct inhibition of E. coli growth at neutral pH al-
though the material chitosan granules showed effective
antibacterial activity only in acidic condition as it has
been known [26,27].
5. Acknowledgements
This work was partially supported by a grant-in-aid for
Industrial Technology Center.
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