International Journal of Food Microbiology 132 (2009) 127133

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Oleoyl-chitosan nanoparticles inhibits Escherichia coli and Staphylococcus aureus

by damaging the cell membrane and putative binding to extracellular
or intracellular targets
Ke Xing a, Xi Guang Chen a,, Cheng Sheng Liu a, Dong Su Cha b, Hyun Jin Park b
a
b

College of Marine Life Science, Ocean University of China, 5# Yushan Road, Qingdao 266003, PR China
The Graduate School of Biotechnology, Korea University, Seoul 136-701, South Korea

a r t i c l e

i n f o

Article history:
Received 25 November 2008
Received in revised form 9 April 2009
Accepted 10 April 2009
Keywords:
Oleoyl-chitosan nanoparticles
Antibacterial dispersion system
Fluorescence

a b s t r a c t
A novel chitosan antibacterial dispersion system was prepared by oleoyl-chitosan (OCS) nanoparticles
(OCNP). We further investigated the antimicrobial mode of OCNP against Escherichia coli and Staphylococcus
aureus using a combination of approaches, including measurement of the effect of lecithin and phosphate
groups, the conformation of membrane protein, internalization of uorescein isothiocyanate (FITC)-labeled
OCS nanoparticles (FITC-OCS nanoparticles) observed under uorescence microscopy and DNA/RNA binding
assay. Results of uorescence experiments indicated that OCNP inuenced the structure of bacterial
membranes. The lecithin effect showed that OCNP bound to cytoplasmic membrane phospholipids of S.
aureus, and phosphate groups played an important role. Fluorescence microscopy observations demonstrated
that the way OCNP entered into bacteria varied against strains. The gel-retardation experiment showed that
OCNP bound strongly to DNA/RNA and retarded their migration in the gels in a concentration-dependent
manner. These results indicate that OCNP exerts its antibacterial activity by damaging the structures of cell
membrane and putative binding to extracellular targets such as phosphate groups or intracellular targets
such as DNA and RNA.
2009 Elsevier B.V. All rights reserved.

1. Introduction
Chitosan is a natural nontoxic biopolymer derived by partially
deacetylated of chitin and had a wide-spectrum antibacterial activity
(Choi et al., 2001; Chung et al., 2003; Liu et al., 2001). Though having
such advantages, chitosan is only soluble in acidic media such as acetic
acid, which also has the antibacterial activity on bacteria. Besides this,
the precipitation occurred upon addition of chitosan solution to the
culture medium, which makes it difcult to investigate the antibacterial activity and antibacterial mechanism of chitosan correctly.
Therefore, in our previous studies (Li et al., 2006, 2007), oleoylchitosan (OCS) nanoparticles (OCNP) were prepared using an O/W
emulsication method based on OCS, which were synthesized by
grafting oleoyl onto the NH2 at C-2 in the chitosan molecule.
Different from chitosan solution, OCNP with strong antibacterial
activity could be well distributed in the culture medium and less
affected by pH of the solution (Xing et al., 2008). These characteristics
make it a novel antibacterial dispersion system of potential value in
the determination of the exact antibacterial mechanism of chitosan.
Until recently, the mechanism of how chitosan acted upon bacteria
has not been elucidated clearly. Different theories have been proposed to
Corresponding author. Tel./fax: +86 532 82032586.
E-mail address: xgchen@ouc.edu.cn (X.G. Chen).
0168-1605/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2009.04.013

explain chitosan's antimicrobial mode of action. In one mechanism

(Helander et al., 2001; Sudarshan et al., 1992), positively charged
chitosan molecules could interfere with the negatively charged residues
on the bacterial surface. Chitosan interacted with the membrane of the
bacteria to alter cell permeability. Other studies (Rabea et al., 2003;
Sudarshan et al., 1992) demonstrated that chitosan with lower
molecular weight might have the intracellular targets. Dissociated
chitosan molecule in solution could bind with DNA and inhibit synthesis
of mRNA through penetration toward the nuclei of the microorganisms
and interfere with the synthesis of mRNA and proteins.
Our previous study (Xing et al., 2009) indicated that OCNP efciently
permeabilized the cell membranes of Escherichia coli and Staphylococcus
aureus. Furthermore, electron microscopy clearly demonstrated OCNP
with intact spherical structure adhered to the bacterial surface, which
supplied direct evidence for the former theory. According to the latter
theory, whether nanoparticles can pass through the cell wall is not
known yet. The lack of understanding of this question led us to our more
systematic study of the mechanism of action.
In this paper, OCNP as a novel antibacterial dispersion system were
prepared. E. coli (Gram-negative) and S. aureus (Gram-positive) were
chosen to be models to elucidate the interaction between OCNP and
bacteria. Effect of lecithin and phosphate groups on the antibacterial
activity and the conformation of membrane protein were investigated.
Internalization of OCNP in bacteria was performed using FITC-OCS

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K. Xing et al. / International Journal of Food Microbiology 132 (2009) 127133

nanoparticles. DNA/RNA binding assay was carried out to gain more

information on OCNP's intracellular targets.
2. Materials and methods
2.1. Materials
Chitosan (MW = 38 kDa), degree of deacetylation 82%, was made
from crab shell and obtained from Biotech (Mokpo, Korea). Fluorescein isothiocyanate (FITC), yolk lecithin and potassium iodide (KI)
were purchased from Sigma Chemicals (Shanghai, China) and used
without further purication.
2.2. Preparation of OCNP
OCNP were prepared according to our previous method (Li et al.,
2006, 2007). In this study, the sample with a degree of substitution
(DS) 5% of oleic acid was chosen to prepare nanoparticles for its
superior antibacterial activity (Xing et al., 2008).
2.3. Cultivation of the microorganisms
E. coli (ATCC 25992) and S. aureus (ATCC 25923) were used as the
test microorganism. A representative bacteria colony was picked up
using a wire loop and placed in nutrient broth (NB, peptone 1%, beef
extract 0.5%, NaCl 0.5%, pH 6), which was then incubated at 37 C for
12 h. By appropriately diluting with sterile nutrient broth, the cultures
of E. coli and S. aureus containing ~107 CFU/mL were prepared and
used for further study.
2.4. Effect of OCNP on uorescence spectra of bacteria membrane protein
Bacterial cultures grown in NB were harvested by centrifugation at
11,000 g for 10 min, washed and resuspended in 0.5% NaCl solution.
The nal cell suspension was adjusted to an absorbance at 610 nm of
0.4. 1 mL bacteria suspension was added into 2 mL potassium iodide
(KI) or OCNP with different concentrations and incubated for 1 h at
110 rpm and 25 C, as mentioned in literatures (Tao et al., 1995; Ye
et al., 2006). The group without KI or OCNP served as the control. The
emission spectra of above samples were scanned from 280 to 500 nm
when both of the slit widths of excitation and emission raster were
5 nm, and the excitation wavelength was xed at 304 nm on an F-4500
uorescence spectrophotometer (Hitachi, Tokyo, Japan).

broth. Two controls were used in this experiment. Control I was

performed with the certain amount of phosphate groups-untreated
OCNP; control II contained only blank culture medium. All of the
samples were inoculated under aseptic conditions with 50 L of the
inocula of bacteria and incubated at 37 C for 24 h, then measured the
turbidity of the cultured medium at 610 nm on a 1601 UVVIS
spectrophotometer (Shimadzu, Tokyo, Japan).
2.6. Fluorescence microscope
2.6.1. Preparation of FITC-OCS
OCS was prepared according to our previous method (Li et al.,
2006). FITC-labeled OCS (FITC-OCS) was synthesized by the reaction
between the isothiocyanate group of FITC and the primary amino
group of OCS (Onishi and Machida, 1999; Wan et al., 2004). Briey,
30 mg OCS was dissolved in 15 mL of 1 M HCl and the pH was adjusted
to 6.9 with 1 M NaOH. FITC (2.1 mg) dissolved in alcohol was added to
the solution above. The mixture was stirred at room temperature for
24 h, and then dialyzed against distilled water using a cellulose
membrane (Sigma, molecular weight cutoff 800010,000) for 2 days,
and lyophilized. All procedures were carried out in the dark. The
synthetic procedure of FITC labeled OCS is shown in Fig. 1.
The FITC content was determined using a 1601 UVVIS spectrophotometer (Shimadzu, Tokyo, Japan) by comparison of the visible
absorption at the maximum wavelength of about 491 nm between the
conjugate and FITC.
2.6.2. Preparation of FITC-OCS nanoparticles
FITC-OCS nanoparticles were prepared according to our previous
method (Li et al., 2007). 30 mg FITC-OCS was dissolved in 15 mL of
0.1 M acetic acid solution. Methylene chloride (3%, v/v) was added to
the FITC-OCS acetic acid solution with stirring and homogenized
(5 min, 16,000 g) for three times with an Ultra-Turrax T-25 dispersing
machine (Kushu Kika Kogyo Co., Osaka, Japan). The solution was held
under vacuum for 30 min at 20 C to remove methylene chloride, and
then sodium tripolyphosphate solution (0.25%, 1 mL) was added as a
cross-linking reagent. The nanoparticle suspension should be prepared and stored in the dark.
2.6.3. Fluorescence microscopy
Bacteria were prepared for uorescence microscopy as described
above. Samples containing E. coli and S. aureus (~107 CFU/mL) in

2.5. Effect of lecithin and phosphate groups on the antibacterial activity

Effect of lecithin on the antibacterial activity was determined as
previously described (Kong et al., 2008). Yolk lecithin was dissolved in
alcohol to prepare the lecithin solution. OCNP were added into the
solution with the mass ratio of 1:0.2, 1:1 and 1:5 to lecithin, respectively.
The mixtures were stirred magnetically overnight, and then were held
under vacuum for 60 min to remove the alcohol. Certain amounts of
OCNP treated with different ratio of lecithin were added to the nutrient
broth. Two controls were used in this experiment. Control I was
performed with the certain amount of lecithin-untreated OCNP; control
II contained only blank culture medium. Test and control groups were
inoculated under aseptic conditions with 50 L of the inocula of bacteria
and incubated at 37 C for 24 h, then measured the turbidity of the
cultured medium at 610 nm on a 1601 UVVIS spectrophotometer
(Shimadzu, Tokyo, Japan).
Effect of phosphate groups on the antibacterial activity was
measured as that of lecithin. Phosphate groups' solutions were prepared
from Na3PO4 aqueous solution and then mixed with OCNP in the mass
ratio of 1:0.2, 1:1 and 1:5, respectively. The mixture was maintained
under magnetic stirring overnight. Certain amounts of OCNP treated
with different ratio of phosphate groups were added to the nutrient

Fig. 1. The synthetic scheme of FITC-labeled OCS.

K. Xing et al. / International Journal of Food Microbiology 132 (2009) 127133

nutrient broth were incubated with 300 mg/L FITC-OCS nanoparticles,

respectively. After incubation in the dark at 37 C for up to 60 min,
the bacteria were pelleted by centrifugation. After washed three
times with phosphate buffered saline, bacteria were resuspended in
phosphate buffer and transferred to slides for observation under a
BX51 uorescence microscopy (Olympus, Tokyo, Japan).

2.7. DNA/RNA binding assay

2.7.1. Preparation of puried E. coli and S. aureus DNA/RNA
E. coli and S. aureus cultures were grown in 50 mL of sterile
nutrient broth at 37 C for 12 h and harvested by centrifugation for
20 min at 8000 g. The supernatant was discarded and 2 mg/mL
lysozyme was added, and the solution was kept at 37 C for 1 h. 20%
sodium-dodecyl-sulfate (SDS) and 20 g/mL proteinase K were added
to the solution, and tubes were vortexed for 1 min and then incubated
at 55 C for 1 h. 550 L phenol-chloroform-isoamyl alcohol (25:24:1)
was added, vortexed, and centrifuged for 10 min at 12,000 g. And the
residual phenol was removed by two extractions with an equal
volume of chloroform-isoamyl alcohol (24:1). 500 L isopropanol and
50 L sodium acetate (3 mol/L, pH 5.2) were added to the aqueous
phase. DNA was precipitated overnight at 20 C. The supernatant
was discarded by centrifugation for 10 min at 12000 g, and 200 L of
70% ethanol was added to the tubes. Tubes were centrifuged for 5 min
at 13,000 rpm at 4 C, and the supernatant was discarded. The pellet
was dried at room temperature then redissolved in 25 L of sterile TE
buffer (10 mM TrisHCl, 1 mM EDTA, pH 8). According to the
manufacturer's instructions, total RNA were extracted from of E. coli
using RNAprep pure Cell/Bacteria Kit (Tiangen, Beijing, China).

2.7.2. Ability of OCNP to binding with DNA/RNA

Gel-retardation experiment was used to test the DNA/RNA
binding abilities of OCNP according to Park et al. (1998). 1 g
genomic DNA or total RNA and OCNP with different concentrations
were mixed and incubated at room temperature for 30 min.
Subsequently, 2 L loading buffer was added and an aliquot of
10 L was applied to 0.8% or 1% agarose gel electrophoresis for DNA
and RNA, respectively.

129

3. Results and discussion

3.1. Effect of OCNP on the membrane protein of bacteria
3.1.1. Determination of excitation peak of Tyr residues on bacterial
membrane protein
There are high contents of proteins in the membranes of bacteria.
Residues in protein, such as tyrosine (Tyr), could emit uorescence
which can be monitored with ultraviolet spectrophotometer. The
more residues in protein exposed in the environment, the stronger the
uorescence intensity will be. On the contrary, the emission
uorescence will be quenched and decreased if the residues count
decrease in the environment. Therefore, the changes in uorescence
spectra of residues in protein could reect the position change of
membrane proteins and the conformation change of cell membranes
indirectly.
The emission peak of Tyr residue was at 303 nm (Tao et al., 1995).
According to the emission wavelength, the excitation peak of Tyr
residue in membrane protein could be determined by scanning at
300800 nm. Results showed that the excitation peak of Tyr residues
was 304 nm for both E. coli and S. aureus.

3.1.2. Effect of uorescence quenching agent on emission peak of Tyr

residues on bacterial membrane protein
KI is a kind of uorescence quenching agent, which can determine
the location of Tyr residues in protein. When uorescence groups
were located on the surface of membrane proteins, I could bind to
these groups directly, causing their emission uorescence to be
quenched and decreased. When uorescence groups were located at
the inside of protein, I could not contact with them, so their
uorescence was not inuenced (Tao et al., 1995; Ye et al., 2007).
Effect of KI on the uorescence emission spectra of the membrane
proteins of E. coli was shown in Fig. 2A. The emission spectra of these
samples were scanned from 280 to 500 nm with a xed excitation
wavelength of 304 nm. KI within the tested concentration quenched
the uorescence of Tyr partially. The uorescence decrease was
greater in suspensions treated with 0.03 mol/L than with 0.015 mol/L
KI. Therefore, the uorescence intensity decrease caused by KI was
dose-dependent. It indicated that part of Tyr residues located on the

Fig. 2. A: Effect of 0.03 mol/L KI ( ), 0.015 mol/L KI () and control () on the uorescence intensity of E. coli membrane protein. The emission spectra of these samples were
scanned from 280 to 500 nm when the excitation wavelength xed at 304 nm. B: Effect of 300 mg/L OCNP ( ), 150 mg/L OCNP () and control () on the uorescence intensity
of E. coli membrane protein. The emission spectra of these samples were scanned from 280 to 500 nm when the excitation wavelength xed at 304 nm.

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surface of the membrane. Therefore, Tyr residues could be used to

reect whether OCNP would interact with the membrane protein.
Effects of KI on the uorescence emission spectra of Try residues of
S. aureus were resemble to that of E. coli (data not shown).
3.1.3. Effect of OCNP on the uorescence intensity of bacterial membrane
protein
When antibacterial agents interacted with cell membrane, the
conformation of membrane proteins would be changed, and Tyr
residues located inside the membrane would be exposed to the surface.
The emission uorescence spectra would be changed correspondingly.
Therefore, changes in the uorescence intensity of the Tyr residue could
sensitively reect whether OCNP would interact with the membrane
protein (Ye et al., 2007).
Effect of OCNP on the uorescence spectra of the membrane
protein of E. coli was shown in Fig. 2B. The uorescence intensity of
Tyr residues increased slightly in OCNP-treated groups. Results
indicated OCNP changed the conformation of membrane protein
and exposed the Tyr residues located inside of membrane, and more
Tyr residues could emit uorescence. Besides this, the uorescence
intensity of Tyr residues increased as the concentration of OCNP
increased from 150 mg/L to 300 mg/L. Results of the uorescence
experiments indicated, OCNP inuenced the structure of cell membranes by interacting with proteins on the cell membrane of the
bacteria. Effects of OCNP on the uorescence emission spectra of Try
residues of S. aureus were resemble to that of E. coli (data not shown).
Therefore, membrane proteins would be one of the target molecules
on cell surfaces for OCNP's action.
3.2. Effect of lecithin on the antibacterial activity of OCNP
Since the cytoplasmic membrane of E. coli and S. aureus primarily
consists of a bi-layer of phospholipid molecules, phospholipid maybe
the targets on the membrane in the interaction between OCNP surface
and the cytoplasmic membrane. Lecithin was involved in this study to
simulate the effect of phospholipids in the cytoplasmic membrane. As
shown in Fig. 3, effect of lecithin on the antibacterial activity of OCNP
was measured by the inhibition rate. For E. coli, OCNP treated with
lecithin of tested concentrations could inhibit bacterial growth
effectively. It meant that lecithin did not inuence the interaction
between OCNP and E. coli. In the case of S. aureus, the addition of
lecithin apparently inuenced the inhibition rate. When the mass
ratio of OCNP to lecithin was 1:0.2, the inhibition rate value decreased
slightly compared with the control group. The value continued to
decrease when the mass ratio of OCNP to lecithin varied to 1:1. When
the mass ratio of OCNP to lecithin varied to 1:5, no marked increase in
viable population was noted with further increase in the content of
lecithin. Results indicated that the antibacterial activity of lecithin-

Fig. 4. Effect of phosphate groups on the antibacterial activity of OCNP against E. coli and
S. aureus. Assays were measured by the inhibition rate. The mass ratio of OCNP/
phosphate-groups was 1:0.2, 1:1 and 1:5. Control was performed with phosphate
groups-untreated OCNP.

treated OCNP against S. aureus was weakened and more viable cells
existed in the suspension. It meant that lecithin inuenced the
interaction between OCNP and S. aureus. Therefore, the effect of
lecithin on the antibacterial activity of OCNP against E. coli and
S. aureus was completely different. In case of S. aureus, phospholipids
might be the target molecules in the interaction which occurred at the
cell surface.
3.3. Effect of phosphate groups on the antibacterial activity
Phosphate groups, the main anionic group existed in cells, play an
important role in the bacterial metabolic activity. For example,
phosphate groups are the composing component of nucleic acids
(DNA and RNA) and phospholipids membrane. Besides this, phosphate groups play a key role in bacterial energy metabolism and
regulation of acidbase balance. To evaluate the possible involvement
of phosphate groups in OCNP's antibacterial activity, we tested E. coli
and S. aureus involved in the antibacterial assay of phosphate groupstreated OCNP.
As shown in Fig. 4, effect of phosphate groups on the antibacterial
activity of OCNP was similar to that of lecithin. For E. coli, phosphate
groups-treated OCNP could inhibit bacterial growth effectively. In the
case of S. aureus, the inhibition rate decreased as the concentration of
phosphate groups increased. Results were agreeable with the
antibacterial assay of lecithin-treated OCNP. Results suggested that,
phosphate groups inuenced the interaction between OCNP and
S. aureus but it was not the case in E. coli.
As essential anionic polymers of the cell walls, phospholipids might
represent a target molecule for OCNP's action against S. aureus, i.e.
phospholipids provided a molecular link for OCNP at the cell surface,
allowing them to disturb membrane functions. As we mentioned above,
phosphate groups are the composing component of phospholipids
membrane. The negatively charged phosphate groups might be an
extracellular target contributing to its interaction with the positively
charged chitosan, ultimately resulting in impairment of vital bacterial
activity. However, we believe that OCNP's mode of action might not
conne to such a single target molecule. Besides the interaction between
OCNP and phosphate groups, there might be other target molecules or
untargeted molecular events. On the basis of our ndings, we believe
that phosphate groups are not the target molecules for OCNP's action
against E. coli. The possible target molecules need to be studied in the
future.
3.4. Fluorescence microscopy

Fig. 3. Effect of lecithin on the antibacterial activity of OCNP against E. coli and S. aureus.
Assays were measured by the inhibition rate. The mass ratio of OCNP/lecithin was 1:0.2,
1:1 and 1:5. Control was performed with lecithin-untreated OCNP.

To search for clues to possible alternative mechanisms of action of

OCNP on E. coli and S. aureus, uorescence microscopy was performed
on bacteria that had been treated with FITC-OCS nanoparticles for up
to 60 min. The uorescence microscopy micrographs showed a
remarkable uorescence of bacterial cell after a period of exposure
to 300 mg/L FITC-OCS nanoparticles.

K. Xing et al. / International Journal of Food Microbiology 132 (2009) 127133

131

Fig. 5. Fluorescence microscopy of E. coli cells after treatment with 300 mg/L FITC-OCS nanoparticles for up to 60 min. A: E. coli treated with FITC-OCS nanoparticles for 5 min;
B: E. coli treated with FITC-OCS nanoparticles for 15 min; C: E. coli treated with FITC-OCS nanoparticles for 30 min; D: E. coli treated with FITC-OCS nanoparticles for 60 min.

When E. coli suspensions were exposed to FITC-OCS nanoparticles

after 5 min (Fig. 5A), dim uorescence was observed. However, the
green uorescence was not distributed inside the bacteria. Instead of
that, the uorescence tends to distribute evenly on the bacterial
surface except the cell poles and the sites of bacterial division. And the
bacterial shape could be faintly discernible. When the incubation time
prolonged to 15 min (Fig. 5B), the uorescence was stronger and more
bacteria labeled with FITC-OCS nanoparticles were visualized. But
the uorescence was still only distributed on the bacterial surface. As
the incubation time increased from 15 min to 30 min (Fig. 5C),
uorescence microscopy analysis conrmed that the intense green
uorescence is indeed the result of FITC-OCS nanoparticles uptake, i.e.
the probe was internalized in FITC-OCS nanoparticles-treated E. coli,
rather than localized on the surface of the bacterial membrane.
Fluorescence microscopy also showed that the FITC-OCS nanoparticles
is not distributed evenly inside the bacteria but instead tends to
cluster in discrete patches. As the arrows pointed to (Fig. 5C), some
interruptions of uorescence in bacteria were clearly observed. When
E. coli suspensions were exposed to FITC-OCS nanoparticles after
60 min (Fig. 5D), uorescence distribute evenly in most of the tested
E. coli cell. As the arrows pointed to, it seemed like there was discrete
distribution of uorescence in a small number of bacteria.
In the case of S. aureus, the uorescence was completely different
from that of E. coli. Bacteria treated with FITC-OCS nanoparticles for as
short as 5 min showed bright green uorescence without discrete
distribution (Fig. 6A). The intense green uorescence was visualized
throughout the tested bacteria, rather than localized on the surface of
the bacterial membrane. As the incubation time increased from 5 min
to 30 min (Fig. 6B and C), the distribution of uorescence intensity
had no signicant change. When S. aureus suspensions were exposed
to FITC-OCS nanoparticles after 60 min (Fig. 6D), many irregular

fragments with bright uorescence were observed, which would be

attributed to the debris of lysed bacteria.
Fluorescence microscopy observation indicated that the ingress of
FITC-OCS nanoparticles varied against E. coli and S. aureus. This was
probably due to the differences in cell wall structure and composition.
The cell wall of E. coli is made up of a thin membrane of peptidoglycan
and an outer membrane composed of lipopolysaccharide, lipoprotein,
and phospholipids. But the peptidoglycan layer of the cell wall of S.
aureus is composed of networks with plenty of pores, so foreign
molecules could enter the cell without difculty. Therefore, it was not
surprising to see that S. aureus was more sensitive than E. coli to FITCOCS nanoparticles.
In our previous study (Xing et al., 2009), electron microscopy
observations evidenced OCNP with intact spherical structure adhering
to the bacterial surface. Results indicated that the membrane-bound
pathway was the rst step ultimately lead to a killing process, which
resulted in apparent holes on the bacterial walls with leakage of
intracellular contents. Meanwhile, the shape of nanoparticles varied
from sphericity to dark occules as the incubation time increased to
30 min (Xing et al., 2009). Mean diameters of nanoparticles were
around 327.4 nm in this antibacterial dispersion system. Chitosan,
being a linear polysaccharide, would have a diameter of around 1.1 nm
in its extended conformation (Dmitriev et al., 2004). However,
chitosan most probably exists in solution in a hydrated form that is
larger than 1.1 nm (Raafat et al., 2008), but still much smaller than the
nanoparticles. Different physical morphology of OCNP and chitosan
determined the different modes they cross the cell wall. Whether
OCNP might be able to pass through the cell wall more easily in the
form of dark occules with smaller fragments? However, this seemed
unlikely in light of the fact that intense green uorescence was
internalized in FITC-OCS nanoparticles treated S. aureus as short as

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K. Xing et al. / International Journal of Food Microbiology 132 (2009) 127133

Fig. 6. Fluorescence microscopy of S. aureus cells after treatment with 300 mg/L FITC-OCS nanoparticles for up to 60 min. A: S. aureus treated with FITC-OCS nanoparticles for 5 min;
B: S. aureus treated with FITC-OCS nanoparticles for 15 min; C: S. aureus treated with FITC-OCS nanoparticles for 30 min; D: S. aureus treated with FITC-OCS nanoparticles for 60 min.

5 min. At this moment, OCNP with intact spherical structure adhered

to the surface of S. aureus in electron micrographs. Therefore, no
evidence would explain how nanoparticles with this size could be able
to cross the cell wall.
3.5. DNA/RNA binding ability of OCNP
The DNA/RNA binding ability of OCNP was examined by analyzing
the effect of OCNP on electrophoretic mobility of bacterial genomic
DNA (Fig. 7) or total RNA (Fig. 8) on agarose gel. As shown in Figs. 7
and 8, the brightness of bands weakened gradually as the concentration of OCNP increased, showing the aggravation of the interactions.
When the concentration achieved 1000 mg/L, OCNP completely
inhibited the migration of E. coli DNA (Fig. 7, lane 6) and RNA (Fig. 8,

Fig. 7. S. aureus DNA binding assay; concentration of OCNP (15): 1000, 500, 300, 150
and 0 mg/L, respectively. E. coli DNA binding assay; concentration of OCNP (610):
1000, 500, 300, 150 and 0 mg/L, respectively.

lane 1). Inhibition of OCNP on electrophoretic mobility of bacterial

genomic DNA or total RNA provided the direct evidence for the
interaction of OCNP with DNA/RNA. As we mentioned above,
phosphate groups might be an extracellular target contributing to
its interaction with the positively charged chitosan, ultimately
resulting in impairment of vital bacterial activity. There are also
phosphate groups with negative charges in the main chain of nucleic
acid (DNA/RNA). The amino groups of the OCNP that possess positive
charges would attract the negatively charged phosphate groups of
DNA/RNA. The possible reason might be that negative charges of
DNA/RNA had been counteracted by OCNP and they would not able to
move in electric eld accordingly, further resulted the weakened
brightness of corresponding bands. Results of electrophoresis pointed
out that the concentration was an important inuencing factor. As the
concentration of OCNP in the medium indicates the concentration of

Fig. 8. E. coli RNA binding assay; concentration of OCNP (610): 1000, 500, 300, 150 and
0 mg/L, respectively.

K. Xing et al. / International Journal of Food Microbiology 132 (2009) 127133

amino groups, results evidenced that the inhibitory effects on bacteria

depended on the amount of amino groups and was strengthened with
the concentration of amino groups in the experimental range (Liu
et al., 2007). The positively charged amino groups of OCNP might be a
fundamental factor contributing to its interaction with the negatively
charged phosphate groups (on the cell surface or intracellular
components), ultimately resulting in impairment of vital bacterial
activities. Investigations on the internalization pathway and other
extracellular or intracellular targets of OCNP are in progress in our
laboratory to nd the exact site of OCNP's action.
4. Conclusions
In this study, we prepared OCNP as a novel antibacterial dispersion
system and investigated the antimicrobial mode of OCNP against
E. coli and S. aureus. The uorescence experiments indicated OCNP
inuenced the structure of membrane and speculated to interact with
proteins on the cell membrane. The lecithin effect suggested that
OCNP bound to cytoplasmic membrane phospholipids of S. aureus,
and phosphate groups might be an extracellular target contributing to
the interaction between OCNP and the cell surface. Fluorescence
microscopy observations demonstrated that the internalization pathway of OCNP was completely different in E. coli and S. aureus. The gelretardation experiment showed that OCNP were capable of binding to
intracellular targets such as DNA and RNA. And the ability was affected
by OCNP's concentration in our research range. These results indicate
that OCNP exerts its antibacterial activity by damaged the structures
of cell membrane and putative binding to extracellular targets such as
phosphate groups or intracellular targets such as DNA and RNA.
Acknowledgements
This work was supported by grants from NSFC (30770582), ISTCP
(2006DFA33150) and the Ph.D. Programs Foundation of Ministry of
Education of China (20070423013).
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